LLP2A-bisphosphonate conjugates for osteoporosis treatment

ABSTRACT

The present invention provides compounds and pharmaceutical compositions of a peptidomimetic ligand, e.g. LLP2A, conjugated with a bisphosphonate drug, e.g. Alendronate. The compounds and pharmaceutical compositions of the present invention are useful in the treatment of osteoporosis and for the promotion of bone growth due to their specificity for the α 4 β 1  integrin on mesenchymal stem cells and for the surface of bone.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a National Stage application under 35 U.S.C. §371 ofInternational Application No. PCT/US12/21909, filed Jan. 19, 2002, whichis a continuation-in-part of International Application No.PCT/US11/50381, filed Sep. 2, 2011, which claims priority to U.S.Provisional Application No. 61/379,643, filed Sep. 2, 2010, which areincorporated in their entirety herein for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with Government support under Grant No. AR057515awarded by the National Institutes of Health. The Government has certainrights in this invention.

REFERENCE TO A SEQUENCE LISTING

The Sequence Listing written in fileSequenceListing_(—)76916-868049.txt, created on Jun. 12, 2015, 8,921bytes, machine format IBM-PC, MS-Windows operating system, is herebyincorporated by reference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

Osteoporosis is a disease of increased bone fragility that results fromestrogen deficiency and aging. It is a major public health problem withnearly 50% of Caucasian women and 25% of Caucasian men at risk for anosteoporotic fracture in their lifetimes (Publication from NationalOsteoporosis Foundation). Accordingly, osteoporosis represents asignificant health concern.

A decrease in the number of mesenchymal stem cells (MSCs) in the bonemarrow with aging leads to reduced osteogenesis and may be the mostimportant factor responsible for reduced bone formation and increasebone fragility (Heersche, J. N., C. G. Bellows, and Y. Ishida, JProsthet Dent, 1998, 79(1): p. 14-6; Ettinger, M. P., Arch Intern Med,2003. 163(18): p. 2237-46). Currently, nearly all of the treatments forosteoporosis reduce bone loss by decreasing osteoclastic bone resorptionand thereby preventing the further breakdown of bone. Importantly, thisclass of drugs does not restore the lost bone structure. Therapeuticmodalities that target bone formation by either increasing the numberand or the activity of osteoblasts may be a more attractive approachthat will enhance bone formation and promote bone regeneration. Althoughbone regeneration by induction of osteogenesis from MSCs is a rationalstrategy to treat osteoporosis, systemic infusions of MSCs in vivo hasfailed to promote an osteogenic response in bone due to the inability ofMSCs to migrate to the bone surface which is a major clinical problemfor MSC transplantation (Gao, J., et al., Cells Tissues Organs, 2001.169(1): p. 12-20; Meyerrose, T. E., et al., Stem Cells, 2007, 25(1): p.220-7). In addition, engraftment of the MSCs requires donor ablationusing chemotherapy and/or radiation which may result in concomitantdamage to endogenous mesenchymal cells (Bacigalupo, A., Best Pract ResClin Haematol, 2004, 17(3): p. 387-99).

Cell adhesion is a process by which cells associate with each other,migrate towards a specific target, or localize within the extracellularmatrix. Cell adhesion constitutes one of the fundamental mechanismsunderlying numerous biological phenomena. Investigations into themolecular basis for cell adhesion have revealed that various cellsurface macromolecules, collectively known as cell adhesion molecules orreceptors, mediate cell-cell and cell-matrix interactions. For example,members of the integrin family of cell surface receptors mediatecell-cell and cell-matrix interactions and regulate cell motility,migration, survival, and proliferation (Hynes, Cell, 69: 11-25 (1992);Hynes, Cell, 1110:673-687 (2002)). Integrins are non-covalentheterodimeric complexes consisting of two subunits, α and β. There areat least 18 different α subunits and at least 8 different β subunits.

Mesenchymal stem cells within the bone marrow have a multi-lineagepotential and represent a mixture of precursors for mesenchymal-derivedcell types including osteoblasts, chondrocytes and adipocytes (Owen, M.et al., Ciba Found Symp, 1988, 136: p. 42-60; Bruder, S. P., et al., JCell Biochem, 1994, 56(3): p. 283-94; Prockop, D. J., Science, 1997,276(5309): p. 71-4). Bone cells at all maturation stages rely heavily oncell-matrix and cell-cell interactions (Mukherjee, S., et al., J ClinInvest, 2008, 118(2): p. 491-504; Grzesik, W. J. and P. G. Robey, J BoneMiner Res, 1994, 9(4): p. 487-96; Vukicevic, S., et al., Cell, 1990,63(2): p. 437-45; Mbalaviele, G., et al., J Bone Miner Res, 2006,21(12): p. 1821-7). Bone marrow is the site where the committedosteoblast progenitors reside, and the osteogenic differentiation is thedefault pathway for MSC lineage commitment (Halleux, C., et al., JMusculoskelet Neuronal Interact, 2001, 2(1): p. 71-6; Muraglia, A., etal., J Cell Sci, 2000, 113 (Pt 7): p. 1161-6). Mobilization of theosteoblastic progenitors to the bone surface is a critical step for theosteoblasts to mature and form mineralized tissue (Adams, G. B., et al.,Nature, 2006, 439(7076): p. 599-603; Chen, X. D., et al., J Bone MinerRes, 2007, 22(12): p. 1943-56). Once the osteoblastic progenitors are“directed” to the bone surface, they synthesize a range of proteinsincluding osteocalcin, osteopontin, bone sialoprotein, osteonectin,collagen-I and fibronectin that will further enhance the adhesion andmaturation of osteoblasts (Gronthos, S., et al., Periodontol 2000, 2006,41: p. 188-95; Gronthos, S., et al., Bone, 2001, 28(2): p. 174-81;Gronthos, S., et al., J Bone Miner Res, 1997, 12(8): p. 1189-97). Theseinteractions are largely mediated by transmembrane integrin receptorsthat primarily utilize an arginine-glycine-aspartate (RGD) sequence toidentify and bind to specific ligands. MSCs express integrins α1, 2, 3,4, 6, 11, CD51 (integrin αV), and CD29 (integrins β1) (Brooke, G., etal., Stem Cells Dev, 2008). Integrins α₁β₁, α₂β₁, α_(v)β₁, α_(v)β₅, α₅β₁and α₄β₁ are reported to be expressed in the osteoblastic cells(Grzesik, W. J. and Robey, P. G., J Bone Miner Res, 1994, 9(4): p.487-96; Gronthos, S., et al., Bone, 2001, 28(2): p. 174-81; Gronthos,S., et al., J Bone Miner Res, 1997. 12(8): p. 1189-97; Cowles, E. A., L.L. Brailey, and G. A. Gronowicz, J Biomed Mater Res, 2000, 52(4): p.725-37). Overexpression of α₄ Integrin on MSCs has been reported toincrease homing of the MSCs to bone (Mukherjee, S., et al., J ClinInvest, 2008, 118(2): p. 491-504).

Bisphosphonates are widely used for the treatment of osteoporosis. Thisclass of drugs is also used as a “vehicle” for delivering bone-targeteddrugs to osseous tissue as prodrugs based on their biphosphonic moiety.Bisphosphonates have been used to deliver sustained release diclofenac,a non-steroidal anti-inflammatory drug to bone in rats (Hirabayashi, H.,et al., J Control Release, 2001, 70(1-2): p. 183-91). The bisphosphonatedose needed for this drug-delivery purpose is usually 10-100 fold lowerthan the doses needed for the treatments of osteoporosis, hypocalcaemia,Paget's disease or metastatic bone cancer.

It is well-understood that bone formation is beneficial for thetreatment of a wide variety of disparate disorders in mammals includingsimple aging, bone degeneration and osteoporosis, fracture healing,fusion or arthrodesis, osteogenesis imperfecta, etc., as well as forsuccessful installation of various medical orthopedic and periodontalimplants such as screws, rods, titanium cage for spinal fusion, hipjoints, knee joint, ankle joints, shoulder joints, dental plates androds, etc.

Increasing bone mineralization to treat conditions characterized atleast in part by increased bone resorption, such as osteopenia, bonefractures, osteoporosis, arthritis, tumor metastases, Paget's diseaseand other metabolic bone disorders, using cathepsin K inhibitors andTGF-beta binding proteins, etc., are well-known as shown by U.S.Publication No. 2004/0235728 to Selwyn Aubrey Stoch, published Nov. 25,2004, and Mary E. Brunkow et al., U.S. Pat. No. 6,489,445 and U.S.Publication No. 2004/0009535, published Jan. 15, 2004. In the Brunkow'445 patent and '535 publication, the TGF-beta binding proteins includeSost polypeptide (full length and short peptide) antibodies thatinterfere with the interaction between the TGF-beta binding proteinsclerostin and a TGF-beta superfamily member, and in particular a bonemorphogenic protein. In the Brunkow '445 patent a novel family of humanTGF-beta binding proteins and nucleic acids encoding them are recited.The protein binds to at least human bone morphogenic protein-5 and humanbone morphogenic protein-6. The aforementioned diseases are due to asystemic loss of bone mineral and thus the administration of theantibody therapeutic is for the systemic (whole body) increase in bonemineral density.

U.S. Publication No. 2006/0165799, published Jul. 27, 2006, teaches abone-filling composition for stimulating bone-formation andbone-consolidation comprising biocompatible calcium sulfate and viscousbiopolymers. The composition is intended to be administered into themissing part of injured bone without diffusing to surrounding organs.

U.S. Publication No. 2005/025604, published Nov. 17, 2005, showsinduction of bone formation by mechanically inducing an increase inosteoblast activity and elevating systemic blood concentration of a boneanabolic agent, including optionally elevating systemic bloodconcentration of an antiresorptive agent.

U.S. Pat. No. 7,576,175, issued Aug. 18, 2009, shows α₄β₁ integrinligands that display high binding affinity, specificity, and stability.The ligands comprise a peptide having n independently selected aminoacids, wherein at least one amino acid is an unnatural amino acid or aD-amino acid, and wherein n is an integer of from 3 to 20.

U.S. Publication No. 2010/0021379, published Jan. 28, 2010, showsantibody conjugates comprising a targeting agent covalently attached toan antibody or fragment thereof. The targeting agent includes a ligandcomprising a peptide or peptidomimetic specific for an integrin receptorsuch as the α₄β₁ integrin.

What is needed in the art is new compositions and methods for treatingosteoporosis and promoting bone growth. Surprisingly, the presentinvention meets these and other needs.

BRIEF SUMMARY OF THE INVENTION

In a first embodiment, the present invention provides a compound ofFormula I:

wherein Formula I each R¹ and R² is independently selected from H,halogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, and C₁₋₆ haloalkyl. R³ is selectedfrom H, C₁₋₆ alkyl and C₃₋₈ cycloalkyl. X is selected from O, S and NH.Y is selected from O and NH. Alternatively, R¹ or R² is combined with Yand the atoms to which they are attached to form a 5-membered heteroarylring. Z is a peptide having 3-20 independently selected amino acids,wherein at least one amino acid is selected from an unnatural amino acidand a D-amino acid. L is a linker. D is a bisphosphonate drug.Subscripts m, n and q are each independently from 0 to 2. Also includedare salts and isomers of the compounds of Formula I.

In a second embodiment, the present invention provides a pharmaceuticalcomposition including a compound of the present invention and apharmaceutically acceptable excipient.

In a third embodiment, the present invention provides a method oftreating osteoporosis, including administering to a subject in needthereof, a therapeutically effective amount of a compound of the presentinvention.

In a fourth embodiment, the present invention provides a method ofpromoting bone growth, including administering to a subject in needthereof, a therapeutically effective amount of a compound of the presentinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows beta-glucuronidase distribution in bone 24-hours followinghuMSCs transplantation in NOD/SCID/MSPVII mice. Three-month-old MSPVIImice received a single intravenous injection of either of PBS, huMSC(5×10⁵), huMSC+LLP2A, or huMSC+LLP2A-Ale. The mice were sacrificed 24hours later. Following sacrifice, lumbar vertebral bodies were harvestedand frozen in Optimal Cutting Temperature embedding media. The sectionswere stained using naphthol-AS-BI-β-Dglucuronide (GUSB) as a substrate.Human MSCs, as showed by GUSB+red stains (black arrows) were seenaccumulated in bone marrow, adjacent to both trabecular and periostealbone surfaces in huMSCs+LLP2A-Ale group.

FIGS. 2 a-2 c show LLP2A-Ale increases trabecular bone formation threeweeks following huMSCs transplantation in NOD/SCID/MSPVII mice. FIG. 2 ashows bone turnover markers measured from the serum. FIG. 2 b showsrepresentative histological sections from the lumber vertebraltrabecular bone from PBS, MSC, MSC+LLP2A or MSC+LLP2A-Ale-treatedgroups. Alizarin red was given at 20 mg/kg at baseline and calcein (10mg/kg) was given subcutaneously 7 days and 2 days before the mice weresacrificed. FIG. 2 c shows surface-based bone histomorphometry performedat the 5^(th) lumbar vertebral bodies three weeks after the injectionand MSC transplantation. ObS/BS, osteoblast surface; MAR, mineralapposition rate; BFR/BS, bone surface-based bone formation rate. *,p<0.05 versus PBS; **, p<0.01 versus PBS. Data are represented asMean±SD.

FIGS. 3 a-3 b show LLP2A-Ale increases cortical bone formation threeweeks following huMSCs transplantation in NOD/SCID/MSPVII mice.Representative cross-sections of the mid-femur from PBS, MSC, MSC+LLP2A,and MSC+LLP2A-Ale-treated groups are shown in FIG. 3 a. Alizarin red wasgiven at 20 mg/kg at baseline (prior to the injection of the studycompound) and calcein (10 mg/kg) was given subcutaneously 7 days and 2days before the mice were sacrificed. Surface-based bonehistomorphometry, performed at the endocortical surface (Ec) and theperiosteal surface (Ps) three weeks after the injections of either MSCsor LLP2A-Ale, is shown in FIG. 3 b. * and ** are as defined above forFIG. 2.

FIGS. 4 a-4 c show LLP2A-Ale increases cancellous bone mass throughincreasing the thickness of the trabeculae. In vivo microCT scans wereperformed on the right distal femurs at baseline (basal) and repeatedafter 4 weeks. Representative 3-dimensional thickness maps from microCTscanning of the cancellous bone from the distal femur metaphysic areshown in FIGS. 4 a-b. The width of the trabeculae is color coded, theblue-green color represents thin trabeculae and yellow-red colorrepresents thick trabeculae. Representative 3-dimensional thickness mapsfrom each group are shown in the lower panel. Percentage changes frombaseline with the different treatments are shown in FIG. 4 c. BV/TVrefers to cancellous bone volume/total tissue volume fraction; Tb.Threfers to trabecular thickness. Maximum load and stress were measured atthe 6^(th) lumbar vertebral body. * and ** are as defined above for FIG.2.

FIGS. 5 a-c show LLP2A-Ale increases cancellous bone formation in young“normal” mice. Bone turnover markers measured from the serum for Runx2and Bglap1 are shown in FIG. 5 a. Bone turnover markers measured fromthe serum for P1NP, osteocalcin, and CTX-1 are shown in FIG. 5 b.Surface-based bone histomorphometry, performed on the right distalfemurs, is shown in FIG. 5 c. ObS/BS, MAR, BFR/BS, *, and ** are asdefined above for FIG. 2.

FIGS. 6 a-b shows LLP2A-Ale increases cortical bone formation in youngnormal mice. Representative cross-sections of the mid-femur from miceinjected with either PBS, LLP2A, Ale, or LLP2A-Ale (10 ng or 250ng/mouse) are shown in FIG. 6 a. Alizarin red was given at 20 mg/kg atbaseline (prior to the injection of the study compound) and calcein (10mg/kg) was given subcutaneously 7 days and 2 days before the mice weresacrificed. Surface-based bone histomorphometry, performed at theendocortical surface (Ec) and the periosteal surface (Ps) four weeksafter the injections, is shown in FIG. 6 b. * and ** are as definedabove for FIG. 2.

FIG. 7 shows osteogenic cells had high affinity for LLP2A-Ale. MouseMSCs were purchased from Invitrogen (Cat#S1502-100), cultured inosteogenic medium for 7 days and incubated with rainbow beads for onehour. The purple beads that displayed LLP2A, the ligand with highaffinity and specificity against α4β1, were covered with layers of cells(green arrows). Original magnification 10× on the right and 20× on theleft of the top panel.

FIG. 8 shows Beta-glucuronidase distribution in various tissues 24-hoursfollowing huMSCs transplantation in NOD/SCID/MSPVII mice.

FIG. 9 shows LLP2A-Ale increases proliferating cell populations adjacentto bone surfaces. Decalcified 4^(th) lumbar vertebral sections weredouble-stained with alkaline phosphatase (blue staining) and BrdU(Bromodeoxyuridine) to monitor the proliferating cells (brown staining,yellow arrows).

FIG. 10 shows the synthetic approach of LLP2A-Ale including FIG. 10A:Synthesis of 4-[(N′-2-methylphenyl)ureido]phenylacetic acid (UPA); FIG.10B: Solid phase synthesis of LLP2A-Lys(D-Cys); FIG. 10C: Preparation ofLLP2A-Ale through conjugating LLP2A-Lys(D-Cys) with Ale-Mal.

FIG. 11 shows the synthesis of LLP2A-Ale (1).

FIG. 12 shows exemplary compounds of the present invention.

FIGS. 13A and B show LLP2A-Ale increases MSC osteoblast maturation,function and migration without affecting their chondrogenic oradipogenic potentials.

FIG. 14 shows (a) retention of the transplanted huMSCs in bone 3-weeksfollowing co-injection in mice with LLP2A-Ale and huMSCs and increasedosteoblasts and osteocytes at (b) cortical and (c) trabecular bongregions in LVB 3-weeks following co-injection in mice with LLP2A-Ale andGFP-labeled huMSCs.

FIG. 15 shows LLP2A-Ale treatment increased osteoblast surface andformed bridges between adjacent trabeculae in immunocompetent mice(129SvJ).

FIGS. 16A and B show bone formation rates on the endocortical surfacesof the tibial shafts were increased in groups that had LLP2A component(P<0.05) in immunocompetent mice (129SvJ).

FIG. 17 shows LLP2A-Ale prevents age-related trabecular bone after peakbone acquisition in C57BL/6 mice.

FIGS. 18A and B show LLP2A-Ale increases bone formation parameters atthe trabecular DF (P<0.05) in C57BL/6 mice.

FIGS. 19A and B shows LLP2A-Ale increases bone formation parameters atthe trabecular bone surfaces in the lumbar vertebral bodies (LVB) inC57BL/6 mice.

FIG. 20 shows scheme of 10-week-old ovariectomized (OVX) mice treatedwith PBS, Ale, LLP2, LLP2A-Ale or PTH 2-weeks after ovariectomy.

FIG. 21 depicts histomorphometric analyses of the 5th lumbar vertebralbodies after LLP2A-Ale treatment of 10-week-old ovariectomized (OVX)mice, showing increases in osteoblast numbers, activities and boneformation rate/bone surface (BFR/BS) post-treatment. Analyses includedtrabecular bone area (%), osteoblast surface (Ob/BS), mineralizingsurface (MS/BS) activities and BFR/BS (P<0.05).

FIG. 22 shows LLP2A-Ale treatment increases osteoblast numbers in thelumbar vertebral bodies (LVB) of 10-week-old ovariectomized (OVX) mice.Representative fluorescent images from the trabecular bone at the 5thLVB (Top panel: white arrowheads illustrate osteoblasts; Bottom panel:yellow arrows illustrate double labeled trabecular bone surfaces).

FIG. 23 depicts histomorphometry analyses of the right mid-femursincluded bone formation at the endosteal (Ec) or periosteal (Ps.) bonesurfaces, and cortical bone thickness of 10-week-old ovariectomized(OVX) mice. Increases in endocortical bone formation from OVX wereobserved in the LLP2A-Ale and PTH treated mice, however cortical bonethickness and maximum stress were not significantly altered by OVX, Ale,and LLP2A, one single IV injection of LLPAle or four weeks of PTHtreatment. Three-point bending was performed on left femurs to obtainmaximum stress of the femurs.

FIG. 24 shows representative fluorescent images from the mid-femursections of 10-week-old ovariectomized (OVX) mice and increases inendocortical bone formation were observed in the LLP2A-Ale and PTHtreatments. Short yellow arrows illustrated double labeled endocorticalbone surfaces. (a, p<0.05 versus Sham; b, P<0.05 vs. OVX+PBS, c, P<0.05vs. OVX+LLP2A; d, P<0.05 vs. OVX+Ale. Data are represented as Mean±SD).

FIG. 25 shows the evaluation of the efficacy of LLP2A-Ale and LLP2A-Ale(1) in their ability in guiding the mesenchymal stem cells (MSC) tobone, we injected 2×105 mouse green florescence labeled (GFP)-MSCs to2-month-old (3-day experiment) or to the 5-month-old (3-week experiment)C57BL/6 mice via I.V. tail vein. LLP2A-Ale or LLP2A-Ale (1) weredissolved in 0.9% normal saline and injected I.P. to the mice one hourafter MSC at a final solution of 0.9 nmol/mouse. Mice were sacrificedeither at 3 days or 3 weeks. Osteocalcin, a bone formation marker, wasmeasured at both 3 days and 3 weeks post-treatments. The lumbarvertebral bodies from the 3-day treatment groups were frozen andsectioned with cryostat. The sections were then stained with anti-GFPantibody. The transplanted MSCs were stained in brown. We found bothLLP2A-Ale and LLP2A-Ale (1) significantly increased osteocalcin levelsat both three-days and three weeks following the treatments.Transplanted MSCs were observed within bone marrow and adjacent to thetrabecular bone surfaces at three-days.

DETAILED DESCRIPTION OF THE INVENTION I. General

The present invention provides compounds and pharmaceutical compositionsof a peptidomimetic ligand, e.g. LLP2A, conjugated with abisphosphonate, e.g. Alendronate. The compounds and pharmaceuticalcompositions of the present invention are useful for the treatment ofosteoporosis, the treatment of low bone mass, the treatment of patientpopulations characterized by fractured or injured bone, and for thepromotion of bone growth due to their specificity for the α₄β₁ integrinon mesenchymal stem cells and for the surface of bone.

II. Definitions

The abbreviations used herein have their conventional meaning within thechemical and biological arts.

As used herein, the terms “Ale” or “Alen”, refer to Alendronate.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents that would result from writing thestructure from right to left, e.g., —CH₂O— is equivalent to —OCH₂—.

As used herein, the terms “halo” or “halogen,” by themselves or as partof another substituent, mean, unless otherwise stated, a fluorine,chlorine, bromine, or iodine atom.

As used herein, the teem “alkyl” refers to a straight or branched,saturated, aliphatic radical having the number of carbon atomsindicated. For example, C₁-C₆ alkyl includes, but is not limited to,methyl, ethyl, propyl, butyl, pentyl, hexyl, iso-propyl, iso-butyl,sec-butyl, tert-butyl, etc.

As used herein, the term “haloalkyl” refers to alkyl as defined abovewhere some or all of the hydrogen atoms are substituted with halogenatoms. For example, haloalkyl includes trifluoromethyl, fluoromethyl,1,2,3,4,5-pentafluoro-phenyl, etc. The term “perfluoro” defines acompound or radical which has at least two available hydrogenssubstituted with fluorine. For example, perfluorophenyl refers to1,2,3,4,5-pentafluorophenyl, perfluoromethane refers to1,1,1-trifluoromethyl, and perfluoromethoxy refers to1,1,1-trifluoromethoxy.

As used herein, the term “heteroalkyl” refers to an alkyl group havingfrom 1 to 3 heteroatoms such as N, O and S. Additional heteroatoms canalso be useful, including, but not limited to B, Al, Si and P. Theheteroatoms can also be oxidized, such as, but not limited to, —S(O)—and —S(O)₂—. For example, heteroalkyl can include ethers, thioethers,alkyl-amines and alkyl-thiols.

As used herein, the term “alkoxy” refers to alkyl with the inclusion ofan oxygen atom, for example, methoxy, ethoxy, etc.

As used herein, the term “aryl” refers to a monocyclic or fusedbicyclic, tricyclic or greater, aromatic ring assembly containing 6 to16 ring carbon atoms. For example, aryl may be phenyl, benzyl ornaphthyl, preferably phenyl. “Arylene” means a divalent radical derivedfrom an aryl group. Aryl groups can be mono-, di- or tri-substituted byone, two or three radicals selected from alkyl, alkoxy, aryl, hydroxy,halogen, cyano, amino, amino-alkyl, trifluoromethyl, alkylenedioxy andoxy-C₂-C₃-alkylene; all of which are optionally further substituted, forinstance as hereinbefore defined; or 1- or 2-naphthyl; or 1- or2-phenanthrenyl. Alkylenedioxy is a divalent substitute attached to twoadjacent carbon atoms of phenyl, e.g. methylenedioxy or ethylenedioxy.Oxy-C₂-C₃-alkylene is also a divalent substituent attached to twoadjacent carbon atoms of phenyl, e.g. oxyethylene or oxypropylene. Anexample for oxy-C₂-C₃-alkylene-phenyl is 2,3-dihydrobenzofuran-5-yl.

Preferred as aryl is phenyl or phenyl mono- or disubstituted by alkyl,heteroalkyl, or halogen.

As used herein, the term “heteroaryl” refers to a monocyclic or fusedbicyclic or tricyclic aromatic ring assembly containing 5 to 16 ringatoms, where from 1 to 4 of the ring atoms are a heteroatom. Forexample, heteroaryl includes pyridyl, indolyl, indazolyl, quinoxalinyl,quinolinyl, isoquinolinyl, benzothienyl, benzofuranyl, furanyl,pyrrolyl, thiazolyl, benzothiazolyl, oxazolyl, isoxazolyl, triazolyl,tetrazolyl, pyrazolyl, imidazolyl, thienyl, or any other radicalssubstituted, especially mono- or di-substituted, by e.g. alkyl, nitro orhalogen. Pyridyl represents 2-, 3- or 4-pyridyl, advantageously 2- or3-pyridyl. Thienyl represents 2- or 3-thienyl. Quinolinyl representspreferably 2-, 3- or 4-quinolinyl. Isoquinolinyl represents preferably1-, 3- or 4-isoquinolinyl. Benzopyranyl, benzothiopyranyl representspreferably 3-benzopyranyl or 3-benzothiopyranyl, respectively. Thiazolylrepresents preferably 2- or 4-thiazolyl, and most preferred,4-thiazolyl. Triazolyl is preferably 1-, 2- or 5-(1,2,4-triazolyl).Tetrazolyl is preferably 5-tetrazolyl.

Preferably, heteroaryl is oxazolyl, imidazolyl, pyridyl, indolyl,quinolinyl, pyrrolyl, thiazolyl, isoxazolyl, triazolyl, tetrazolyl,pyrazolyl, imidazolyl, thienyl, furanyl, benzothiazolyl, benzofuranyl,isoquinolinyl, benzothienyl, indazolyl, or any of the radicalssubstituted, especially mono- or di-substituted.

Substituents for the aryl and heteroaryl groups are varied and areselected from: -halogen, —OR′, —OC(O)R′, —NR′R″, —SR′, —R′, —CN, —NO₂,—CO₂R′, —CONR′R″, —C(O)R′, —OC(O)NR′R″, —NR″C(O)R′, —NR″C(O)₂R′,—NR′—C(O)NR″R′″, —NH—C(NH₂)═NH, —NR′C(NH₂)═NH, —NH—C(NH₂)═NR′, —S(O)R′,—S(O)₂R′, —S(O)₂NR′R″, —N₃, —CH(Ph)₂, perfluoro(C₁-C₄)alkoxy, andperfluoro(C₁-C₄)alkyl, in a number ranging from zero to the total numberof open valences on the aromatic ring system; and where R′, R″ and R′″are independently selected from hydrogen, C₁-C₈ alkyl and heteroalkyl,unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C₁-C₄)alkyl,and (unsubstituted aryl)oxy-(C₁-C₄)alkyl.

As used herein, the term “cycloalkyl” refers to a saturated or partiallyunsaturated, monocyclic, fused bicyclic or bridged polycyclic ringassembly containing from 3 to 12 ring atoms, or the number of atomsindicated. For example, C₃₋₈cycloalkyl includes cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, and up to cyclooctyl.

As used herein, each of the above terms (e.g., “alkyl,” “aryl” and“heteroaryl”) are meant to include both substituted and unsubstitutedforms of the indicated radical.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NR(SO₂)R′, —CN and—NO₂ in a number ranging from zero to (2 m′+1), where m′ is the totalnumber of carbon atoms in such radical. R′, R″, R′″ and R″″ are eachindependently selected from hydrogen, C₁-C₈ alkyl and heteroalkyl,unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C₁-C₄)alkyl,and (unsubstituted aryl)oxy-(C₁-C₄)alkyl. When a compound of theinvention includes more than one R group, for example, each of the Rgroups is independently selected as are each R′, R″, R′″ and R″″ groupswhen more than one of these groups is present. When R′ and R″ areattached to the same nitrogen atom, they can be combined with thenitrogen atom to form a 4-, 5-, 6-, or 7-membered ring. For example,—NR′R″ is meant to include, but not be limited to, 1-pyrrolidinyl and4-morpholinyl. From the above discussion of substituents, one of skillin the art will understand that the term “alkyl” is meant to includegroups including carbon atoms bound to groups other than hydrogengroups, such as haloalkyl (e.g., —CF₃ and —CH₂CF₃) and acyl (e.g.,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

As used herein, the term “peptide” refers to a compound made up of asingle chain of D- or L-amino acids or a mixture of D- and L-amino acidsjoined by peptide bonds. Generally, peptides are about 2 to about 50amino acids in length. Preferably, the peptides of the present inventionare about 2 to about 25 amino acids in length, more preferably 3 to 20amino acids in length, and most preferably 3 to 10 amino acids inlength.

As used herein, the term “amino acid” refers to naturally occurring,unnatural, and synthetic amino acids, as well as amino acid analogs andamino acid mimetics that function in a manner similar to the naturallyoccurring amino acids.

As used herein, the terms “naturally-occurring amino acids” refer tothose amino acids which are encoded by the genetic code, as well asthose amino acids that are later modified, e.g., hydroxyproline,γ-carboxyglutamate and O-phosphoserine. Naturally-occurring α-aminoacids include, without limitation, alanine (Ala), cysteine (Cys),aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine(Gly), histidine (His), isoleucine (Ile), arginine (Arg), lysine (Lys),leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro),glutamine (Gln), serine (Ser), threonine (Thr), valine (Val), tryptophan(Trp), tyrosine (Tyr), and combinations thereof. Stereoisomers of anaturally-occurring α-amino acids include, without limitation, D-alanine(D-Ala), D-cysteine (D-Cys), D-aspartic acid (D-Asp), D-glutamic acid(D-Glu), D-phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine(D-Ile), D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu),D-methionine (D-Met), D-asparagine (D-Asn), D-proline (D-Pro),D-glutamine (D-Gln), D-serine (D-Ser), D-threonine (D-Thr), D-valine(D-Val), D-tryptophan (D-Trp), D-tyrosine (D-Tyr), and combinationsthereof.

As used herein, the terms “unnatural amino acids” include, withoutlimitation, amino acid analogs, amino acid mimetics, synthetic aminoacids, N-substituted glycines, and N-methyl amino acids in either the L-or D-configuration that function in a manner similar to thenaturally-occurring amino acids. Unnatural amino acids” are not encodedby the genetic code and can, but do not necessarily have the same basicstructure as a naturally occurring amino acid.

As used herein, the terms “amino acid analogs” refer to compounds thathave the same basic chemical structure as a naturally occurring aminoacid, i.e., an α carbon that is bound to a hydrogen, a carboxyl group,an amino group, and an R group, e.g., homoserine, norleucine, methioninesulfoxide, methionine methyl sulfonium. Such analogs have modified Rgroups (e.g., norleucine) or modified peptide backbones, but retain thesame basic chemical structure as a naturally occurring amino acid.

As used herein, the tetras “amino acid mimetics” refer to chemicalcompounds that have a structure that is different from the generalchemical structure of an amino acid, but that function in a mannersimilar to a naturally-occurring amino acid. Suitable amino acidmimetics include, without limitation, β-amino acids and γ-amino acids.In β-amino acids, the amino group is bonded to the β-carbon atom of thecarboxyl group such that there are two carbon atoms between the aminoand carboxyl groups. In γ-amino acids, the amino group is bonded to theγ-carbon atom of the carboxyl group such that there are three carbonatoms between the amino and carboxyl groups. Suitable R groups for β- orγ-amino acids include, but are not limited to, side-chains present innaturally-occurring amino acids and unnatural amino acids.

As used herein, the terms “N-substituted glycines” refer to unnaturalamino acids based on glycine, where an amino acid side-chain is attachedto the glycine nitrogen atom. Suitable amino acid side-chains (e.g., Rgroups) include, but are not limited to, side chains present innaturally-occurring amino acids and side-chains present in unnaturalamino acids such as amino acid analogs.

Amino acids can be characterized by at least one of several properties.For example, amino acids can be positively charged, negatively charged,hydrophilic, or hydrophobic.

As used herein, the terms “positively charged amino acid” refer to thoseamino acids having a basic or positively charged side chain at pH valuesbelow the pKa, and include, but are not limited to, Lys, Arg, HoArg,Agp, Agb, Dab, Dap and Orn and stereoisomers thereof. Basic amino acidscan generally be referred to by the symbol “X⁺”.

As used herein, the terms “negatively charged amino acid” refer to thoseamino acids having an acidic or negatively charged side chain at pHvalues above the pKa, and include, but are not limited to, Asp, Glu,Aad, Bec and stereoisomers thereof. Acidic amino acids can generally bereferred to by the symbol “X⁻”. One of skill in the art will appreciatethat other basic and acidic amino acids are known in the art.

As used herein, the terms “neutrally charged amino acids” refer to thoseamino acids having a neutrally charged side chain at pH values equal tothe pKa.

As used herein, the terms “hydrophilic amino acid” refer to those aminoacids having a polar and uncharged side chain and include, but are notlimited to, Asn, Ser, Thr, and Gln.

As used herein, the terms “hydrophobic amino acids” refer to those aminoacids having a hydrophobic side chain and include, but are not limitedto, Val, Leu, Ile, Met, and Phe. Preferably, the hydrophobic amino acidis selected from proline, a proline analog, and a stereoisomer thereof.Preferably, the proline analog is hydroxyproline.

As used herein, the terms “D-amino acids” refer to the D stereoisomer ofan amino acid. The letters D and L are conventionally used in the art torefer to the stereoisomers of an amino acid. D-amino acids are thoseamino acids that could be synthesized from the dextrorotary isomer ofglyceraldehyde, i.e. D-glyceraldehyde. Similarly, L-amino acids arethose amino acids that could be synthesized from the levorotary isomerof glyceraldehyde, i.e. L-glyceraldehyde.

Amino acids may be referred to herein by either the commonly known threeletter symbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission. Nucleotides, likewise, may bereferred to by their commonly accepted single-letter codes.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid(i.e., hydrophobic, hydrophilic, positively charged, neutral, negativelycharged). The chemically similar amino acid includes, withoutlimitation, a naturally-occurring amino acid such as an L-amino acid, astereoisomer of a naturally occurring amino acid such as a D-amino acid,and an unnatural amino acid such as an amino acid analog, amino acidmimetic, synthetic amino acid, N-substituted glycine, and N-methyl aminoacid. Exemplified hydrophobic amino acids include valine, leucine,isoleucine, methionine, phenylalanine, and tryptophan. Exemplifiedaromatic amino acids include phenylalanine, tyrosine and tryptophan.Exemplified aliphatic amino acids include serine and threonine.Exemplified basic amino acids include lysine, arginine and histidine.Exemplified amino acids with carboxylate side-chains include aspartateand glutamate. Exemplified amino acids with carboxamide side chainsinclude asparagines and glutamine. Conservative substitution tablesproviding functionally similar amino acids are well known in the art.Such conservatively modified variants are in addition to and do notexclude polymorphic variants, interspecies homologs, and alleles of theinvention.

Conservative substitution tables providing functionally similar aminoacids are well known in the art. For example, substitutions may be madewherein an aliphatic amino acid (e.g., G, A, I, L, or V) is substitutedwith another member of the group. Similarly, an aliphaticpolar-uncharged group such as C, S, T, M, N, or Q, may be substitutedwith another member of the group; and basic residues, e.g., K, R, or H,may be substituted for one another. In some embodiments, an amino acidwith an acidic side chain, e.g., E or D, may be substituted with itsuncharged counterpart, e.g., Q or N, respectively; or vice versa. Eachof the following eight groups contains other exemplary amino acids thatare conservative substitutions for one another:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

7) Serine (S), Threonine (T); and

8) Cysteine (C), Methionine (M)

(see, e.g., Creighton, Proteins (1984)).

As used herein, the term “linker” refers to a moiety that possesses oneor more different reactive functional groups that allows for covalentattachment of moieties such as a peptide to a chelating agent.Preferably, the linking moiety possesses two different reactivefunctional groups, i.e., a heterobifunctional linker. Suitable linkersinclude, without limitation, those available from Pierce Biotechnology,Inc. (Rockford, Ill.). In preferred embodiments of the presentinvention, the linker provides a carboxyl group for the attachment of achelating agent and an amino group for the attachment of a peptide.However, one skilled in the art understands that any reactive functionalgroup can be present on the linker, as long as it is compatible with afunctional group on the moiety that is to be covalently attached.

Linkers useful in the present invention includes those possessing one ormore different reactive functional groups that allow for covalentattachment of moieties such as a peptide to a chelating agent. Thelinking moiety possesses two or more different reactive functionalgroups. In some cases multivalent linkers can be used. Suitable linkersinclude, without limitation, those available from Pierce Biotechnology,Inc. (Rockford, Ill.). In preferred embodiments of the presentinvention, the linker provides a carboxyl group for the attachment of achelating agent and an amino group for the attachment of a peptide.However, one skilled in the art understands that any reactive functionalgroup can be present on the linker, as long as it is compatible with afunctional group on the moiety that is to be covalently attached. Asused herein, the term “chelating agent-linker conjugate” refers to achelating agent covalently attached to a linker. Such chelatingagent-linker conjugates can be attached to a peptide via a functionalgroup present on the linker. Some suitable linkers include, but are notlimited to, β-alanine, 2,2′-ethylenedioxy bis(ethylamine)monosuccinamide(Ebes) and bis(Ebes)-Lys. Other suitable linkers include those withbiotin. Additional linkers can be found in Bioconjugate Techniques, GregT. Hermanson, Academic Press, 2d ed., 2008 (incorporated by reference inits entirety herein).

As used herein, the term “salt” refers to acid or base salts of thecompounds used in the methods of the present invention. Illustrativeexamples of pharmaceutically acceptable salts are mineral acid(hydrochloric acid, hydrobromic acid, phosphoric acid, and the like)salts, organic carboxylic acids (acetic acid, propionic acid, glutamicacid, citric acid and the like), organic sulfonic acids (methanesulfonicacid), salts, quaternary ammonium (methyl iodide, ethyl iodide, and thelike) salts. It is understood that the pharmaceutically acceptable saltsare non-toxic. Additional information on suitable pharmaceuticallyacceptable salts can be found in Remington's Pharmaceutical Sciences,17th ed., Mack Publishing Company, Easton, Pa., 1985, which isincorporated herein by reference.

Pharmaceutically acceptable salts of the acidic compounds of the presentinvention include salts formed with bases, namely cationic salts such asalkali and alkaline earth metal salts, such as sodium, lithium,potassium, calcium, magnesium, as well as ammonium salts, such asammonium, trimethyl-ammonium, diethylammonium, andtris-(hydroxymethyl)-methyl-ammonium salts.

As used herein, the term “hydrate” refers to a compound that iscomplexed to at least one water molecule. The compounds of the presentinvention can be complexed with from 1 to 10 water molecules.

As used herein, the terms “pharmaceutically acceptable excipient” and“pharmaceutically acceptable carrier” refer to a substance that aids theadministration of an active agent to and absorption by a subject.“Pharmaceutically acceptable excipient” refers to an excipient that canbe included in the compositions of the invention and that causes nosignificant adverse toxicological effect on the patient. Non-limitingexamples of pharmaceutically acceptable excipients include water, NaCl,normal saline solutions, lactated Ringer's, normal sucrose, normalglucose, binders, fillers, disintegrants, lubricants, coatings,sweeteners, flavors and colors, and the like. One of skill in the artwill recognize that other pharmaceutical excipients are useful in thepresent invention.

As used herein, the term “osteoporosis” refers to a disease of increasedbone fragility that results from estrogen deficiency and aging.

As used herein, the term “isomers” refers to compounds with the samechemical formula but which are structurally distinguishable.

As used herein, the term “tautomer,” refers to one of two or morestructural isomers which exist in equilibrium and which are readilyconverted from one isomeric form to another.

As used herein, the terms “patient” or “subject” refers to a livingorganism suffering from or prone to a condition that can be treated byadministration of a pharmaceutical composition as provided herein.Non-limiting examples include humans, other mammals and othernon-mammalian animals.

As used herein, the term “therapeutically effective amount” refers to anamount of a conjugated functional agent or of a pharmaceuticalcomposition useful for treating or ameliorating an identified disease orcondition, or for exhibiting a detectable therapeutic or inhibitoryeffect. The effect can be detected by any assay method known in the art.

As used herein, the terms “treat”, “treating” and “treatment” refers toany indicia of success in the treatment or amelioration of an injury,pathology or condition, including any objective or subjective parametersuch as abatement; remission; diminishing of symptoms or making theinjury, pathology or condition more tolerable to the patient; slowing inthe rate of degeneration or decline; making the final point ofdegeneration less debilitating; improving a patient's physical or mentalwell-being. The treatment or amelioration of symptoms can be based onobjective or subjective parameters; including the results of a physicalexamination, neuropsychiatric exams, and/or a psychiatric evaluation.For example, the methods of the invention successfully treat a patient'sdelirium by decreasing the incidence of disturbances in consciousness orcognition.

As used herein, the terms “disorder” or “condition” refer to a state ofbeing or health status of a patient or subject capable of being treatedwith the glucocorticoid receptor modulators of the present invention.

As used herein, the terms “a,” “an,” or “a(n)”, when used in referenceto a group of substituents or “substituent group” herein, mean at leastone. For example, where a compound is substituted with “an” alkyl oraryl, the compound is optionally substituted with at least one alkyland/or at least one aryl, wherein each alkyl and/or aryl is optionallydifferent. In another example, where a compound is substituted with “a”substituent group, the compound is substituted with at least onesubstituent group, wherein each substituent group is optionallydifferent.

Description of compounds of the present invention is limited byprinciples of chemical bonding known to those skilled in the art.Accordingly, where a group may be substituted by one or more of a numberof substituents, such substitutions are selected so as to comply withprinciples of chemical bonding and to give compounds which are notinherently unstable and/or would be known to one of ordinary skill inthe art as likely to be unstable under ambient conditions, such asaqueous, neutral, or physiological conditions.

III. Compounds

In some embodiments, the present invention provides a compound ofFormula I:

wherein Formula I each R¹ and R² is independently selected from H,halogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, and C₁₋₆ haloalkyl. R³ is selectedfrom H, C₁₋₆ alkyl and C₃₋₈ cycloalkyl. X is selected from O, S and NH.Y is selected from O and NH. Alternatively, R¹ or R² is combined with Yand the atoms to which they are attached to form a 5-membered heteroarylring. Z is a peptide having 3-20 independently selected amino acids,wherein at least one amino acid is selected from an unnatural amino acidand a D-amino acid. L is a linker. D is a bisphosphonate drug.Subscripts m, n and q are each independently from 0 to 2. Also includedare the salts and isomers of the compounds of formula I.

C₃₋₈ cycloalkyl groups useful with the present invention include, butare not limited to, cyclopropane, cyclobutane, cyclopentane,cyclohexane, cycloheptane, and cyclooctane. Preferably, C₃₋₈ cycloalkylgroups include cyclohexane. 5-membered heteroaryl groups useful with thepresent invention, include, but are not limited to imidazole, oxazole,isoxazole, thiazole, and isothiazole.

Amino acids useful with the present invention include, withoutlimitation, naturally occurring amino acids; D-amino acids; unnaturalamino acids which include, without limitation, amino acid analogs, aminoacid mimetics, N-substituted glycines, N-methyl amino acids,phenylalanine analogs, and derivatives of lysine (Lys), ornithine (Orn)and α,γ-diaminobutyric acid (Dbu) in either the L- or D-configuration;hydrophilic amino acids, hydrophobic amino acids; and negatively chargedamino acids.

Naturally-occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate and O-phosphoserine.Naturally-occurring α-amino acids include, without limitation, alanine(Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu),phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile),arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met),asparagine (Asn), proline (Pro), glutamine (Gln), serine (Ser),threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), andcombinations thereof.

Stereoisomers of a naturally-occurring α-amino acids include, withoutlimitation, D- and L-amino acids. D-amino acids suitable for use in thepresent invention include, without limitation, D-alanine (D-Ala),D-cysteine (D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu),D-phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D-Ile),D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu), D-methionine(D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln),D-serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D-tryptophan(D-Trp), D-tyrosine (D-Tyr), and combinations thereof. In some otherembodiments, the D-amino acid is selected from a D-α-amino acid, aD-β-amino acid, a D-γ-amino acid, and a combination thereof. In yetanother embodiment, the D-α-amino acid is selected from a stereoisomerof a naturally-occurring α-amino acid, an unnatural D-α-amino acid, anda combination thereof.

Unnatural amino acids include, without limitation, amino acid analogs,amino acid mimetics, N-substituted glycines, N-methyl amino acids,phenylalanine analogs, and derivatives of lysine (Lys), ornithine (Orn)and α,γ-diaminobutyric acid (Dbu) in either the L- or D-configurationthat function in a manner similar to the naturally-occurring aminoacids. Unnatural amino acids are not encoded by the genetic code, andcan, but do not necessarily, have the same basic structure or functionas a naturally occurring amino acid.

Unnatural amino acids useful with the present invention include, but arenot limited to azetidinecarboxylic acid, 2-aminoadipic acid,3-aminoadipic acid, beta-alanine, aminopropionic acid, 2-aminobutyricacid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid,2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid,tertiary-butylglycine, 2,4-diaminoisobutyric acid, desmosine,2,2′-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine,N-ethylasparagine, homoproline, hydroxylysine, allo-hydroxylysine,3-hydroxyproline, 4-hydroxyproline, isodemosine, allo-isoleucine,N-methylalanine, N-methylglycine, N-methylisoleucine,N-methylpentylglycine, N-methylvaline, naphthalanine, norvaline,ornithine, pentylglycine, pipecolic acid, thioproline,aminophenylalanine, hydroxytyrosine, and aminotyrosine.

In some other embodiments, the unnatural amino acid is selected from1-aminocyclopentane-1-carboxylic acid (Acp),1-aminocyclobutane-1-carboxylic acid (Acb),1-aminocyclopropane-1-carboxylic acid (Acpc), citrulline (Cit),homocitrulline (HoCit), α-aminohexanedioic acid (Aad),3-(4-pyridyl)alanine (4-Pal), 3-(3-pyridyl)alanine (3-Pal),propargylglycine (Pra), α-aminoisobutyric acid (Aib), α-aminobutyricacid (Abu), norvaline (Nva), α,β-diaminopropionic acid (Dpr),α,γ-diaminobutyric acid (Dbu), α-tert-butylglycine (Bug),3,5-dinitrotyrosine Tyr(3,5-di NO₂), norleucine (Nle),3-(2-naphthyl)alanine (Nal-2), 3-(1-naphthyl)alanine (Nal-1),cyclohexylalanine (Cha), di-n-propylglycine (Dpg), cyclopropylalanine(Cpa), homoleucine (Hle), homoserine (HoSer), homoarginine (Har),homocysteine (Hcy), methionine sulfoxide (Met(O)), methioninemethylsulfonium (Met (S-Me)), α-cyclohexylglycine (Chg),3-benzo-thienylalanine (Bta), taurine (Tau), hydroxyproline (Hyp),O-benzyl-hydroxyproline (Hyp(Bzl)), homoproline (HoPro), β-homoproline(βHoro), thiazolidine-4-carboxylic acid (Thz), nipecotic acid (Nip),isonipecotic acid (IsoNip),3-carboxymethyl-1-phenyl-1,3,8-triazaspiro[4,5]decan-4-one (Cptd),tetrahydro-isoquinoline-3-carboxylic acid (3-Tic),5H-thiazolo[3,2-a]pyridine-3-carboxylic acid (Btd), 3-aminobenzoic acid(3-Abz), 3-(2-thienyl)alanine (2-Thi), 3-(3-thienyl)alanine (3-Thi),α-aminooctanedioc acid (Asu), diethylglycine (Deg),4-amino-4-carboxy-1,1-dioxo-tetrahydrothiopyran (Acdt),1-amino-1-(4-hydroxycyclohexyl)carboxylic acid (Ahch),1-amino-1-(4-ketocyclohexyl)carboxylic acid (Akch),4-amino-4-carboxytetrahydropyran (Actp), 3-nitrotyrosine (Tyr(3-NO₂)),1-amino-1-cyclohexane carboxylic acid (Ach),1-amino-1-(3-piperidinyl)carboxylic acid (3-Apc),1-amino-1-(4-piperidinyl)carboxylic acid (4-Apc),2-amino-3-(4-piperidinyl)propionic acid (4-App),2-aminoindane-2-carboxylic acid (Aic), 2-amino-2-naphthylacetic acid(Ana), (2S,5R)-5-phenylpyrrolidine-2-carboxylic acid (Ppca),4-thiazoylalanine (Tha), 2-aminooctanoic acid (Aoa), 2-aminoheptanoicacid (Aha), ornithine (Orn), azetidine-2-carboxylic acid (Aca),α-amino-3-chloro-4,5-dihydro-5-isoazoleacetic acid (Acdi),thiazolidine-2-carboxylic acid (Thz(2-COOH)), allylglycine (Agl),4-cyano-2-aminobutyric acid (Cab), 2-pyridylalanine (2-Pal),2-quinoylalanine (2-Qal), cyclobutylalanine (Cba), a phenylalanineanalog, a lysine derivative, a ornithine (Orn) derivative, anα,γ-diaminobutyric acid Dbu derivative, stereoisomers thereof, andcombinations thereof (see, Liu and Lam, Anal. Biochem., 295:9-16(2001)). As such, the unnatural α-amino acids are present either asunnatural L-α-amino acids, unnatural D-α-amino acids, or combinationsthereof.

In some embodiments, the unnatural amino acids is selected fromcompounds of Table 1:

TABLE 1 Unnatural amino acids useful with the present invention.Fmoc-Aad(OtBu)-OH Fmoc-Bec(OtBu)-OH Fmoc-Bmc(OtBu)-OH Fmoc-Ach-OH

Suitable amino acid analogs include, without limitation, homoserine,norleucine, methionine sulfoxide, methionine methyl sulfonium.

Suitable amino acid mimetics include, without limitation, β-amino acidsand γ-amino acids. Suitable R groups for β- or γ-amino acids include,but are not limited to, side-chains present in naturally-occurring aminoacids and unnatural amino acids.

N-substituted glycines suitable for use in the present inventioninclude, without limitation, N-(2-aminoethyl)glycine,N-(3-aminopropyl)glycine, N-(2-methoxyethyl)glycine, N-benzylglycine,(S)-N-(1-phenylethyl)glycine, N-cyclohexylmethylglycine,N-(2-phenylethyl)glycine, N-(3-phenylpropyl)glycine,N-(6-aminogalactosyl)glycine, N-(2-(3′-indolylethyl)glycine,N-(2-(p-methoxyphenylethyl))glycine, N-(2-(p-chlorophenylethyl)glycine,and N-[2-(p-hydroxyphenylethyl)]glycine. N-substituted glycineoligomers, referred to herein as “peptoids,” have been shown to beprotease resistant (Miller et al., Drug Dev. Res., 35:20-32 (1995)). Assuch, peptoids containing at least one unnatural α-amino acid, D-aminoacid, or a combination thereof are within the scope of the presentinvention.

Suitable N-methyl amino acids include N-methyl-Ala, N-methyl-Cys,N-methyl-Asp, N-methyl-Glu, N-methyl-Phe, N-methyl-Gly, N-methyl-His,N-methyl-Ile, N-methyl-Arg, N-methyl-Lys, N-methyl-Leu, N-methyl-Met,N-methyl-Asn, N-methyl-Gln, N-methyl-Ser, N-methyl-Thr, N-methyl-Val,N-methyl-Trp, N-methyl-Tyr, N-methyl-Acp, N-methyl-Acb, N-methyl-Acpc,N-methyl-Cit, N-methyl-HoCit, N-methyl-Aad, N-methyl-4-Pal,N-methyl-3-Pal, N-methyl-Pra, N-methyl-Aib, N-methyl-Abu, N-methyl-Nva,N-methyl-Dpr, N-methyl-Dbu, N-methyl-Nle, N-methyl-Nal-2,N-methyl-Nal-1, N-methyl-Cha, N-methyl-Cpa, N-methyl-Hle,N-methyl-HoSer, N-methyl-Har, N-methyl-Hcy, N-methyl-Chg, N-methyl-Bta,N-methyl-2-Thi, N-methyl-3-Thi, N-methyl-Asu, N-methyl-Acdt,N-methyl-Ahch, N-methyl-Akch, N-methyl-Actp, N-methyl-Tyr(3-NO₂),N-methyl-Ach, N-methyl-3-Apc, N-methyl-4-Apc, N-methyl-4-App,N-methyl-Tha, N-methyl-Aoa, N-methyl-Aha, N-methyl-Orn, N-methyl-Aca,N-methyl-Agl, N-methyl-Cab, N-methyl-2-Pal, N-methyl-Cba,N-methyl-HoPhe, N-methyl-Phg, N-methyl-Phe(4-NH₂), N-methyl-4-Phe(4-Me),N-methyl-Phe(4-F), N-methyl-Phe(4-Cl), N-methyl-Phe(2-Br),N-methyl-Phe(3-Br), N-methyl-Phe(4-Br), N-methyl-Phe(3-CF₃),N-methyl-Phe(4-CF₃), N-methyl-Phe(4-NO₂), N-methyl-Phe(4-CN),N-methyl-Bpa, N-methyl-Phg(4-Cl), N-methyl-Phg(4-Br), N-methyl-Tyr(Me),N-methyl-Lys38, N-methyl-Lys27, N-methyl-Lys73, N-methyl-Lys55,N-methyl-Lys28, N-methyl-Lys72, N-methyl-Lys12, N-methyl-Lys123,N-methyl-Lys63, N-methyl-Lys124, N-methyl-Lys82, N-methyl-Lys31,N-methyl-Lys15, N-methyl-Lys125, N-methyl-Lys43, N-methyl-Lys24,N-methyl-Lys5, N-methyl-Lys4, N-methyl-Lys50, N-methyl-Lys81,N-methyl-Orn38, N-methyl-Orn27, N-methyl-Orn73, N-methyl-Orn55,N-methyl-Orn28, N-methyl-Orn72, N-methyl-Orn12, N-methyl-Orn123,N-methyl-Orn63, N-methyl-Orn124, N-methyl-Orn82, N-methyl-Orn31,N-methyl-Orn15, N-methyl-Orn125, N-methyl-Orn43, N-methyl-Orn24,N-methyl-Orn5, N-methyl-Orn4, N-methyl-Orn50, N-methyl-Orn81,N-methyl-Dbu38, N-methyl-Dbu27, N-methyl-Dbu73, N-methyl-Dbu55,N-methyl-Dbu28, N-methyl-Dbu72, N-methyl-Dbu12, N-methyl-Dbu123,N-methyl-Dbu63, N-methyl-Dbu124, N-methyl-Dbu82, N-methyl-Dbu31,N-methyl-Dbu15, N-methyl-Dbu125, N-methyl-Dbu43, N-methyl-Dbu24,N-methyl-Dbu5, N-methyl-Dbu4, N-methyl-Dbu50, N-methyl-Dbu81,stereoisomers thereof, and combinations thereof.

Suitable phenylalanine analogs useful with the present inventioninclude, without limitation, homophenylalanine (HoPhe), phenylglycine(Phg), 3,3-diphenylalanine (Dpa), 4-aminophenylalanine (Phe(4-NH₂)),2-methylphenylalanine (Phe(2-Me)), 3-methylphenylalanine (Phe(3-Me)),4-methylphenylalanine (Phe(4-Me)), 4-azidophenylalanine (Phe(4-N₃)),2-fluorophenylalanine (Phe(2-F)), 3-fluorophenylalanine (Phe(3-F)),4-fluorophenylalanine (Phe(4-F)), 2-chlorophenylalanine (Phe(2-Cl)),3-chlorophenylalanine (Phe(3-Cl)), 4-chlorophenylalanine (Phe(4-Cl)),2-bromophenylalanine (Phe(2-Br)), 3-bromophenylalanine (Phe(3-Br)),4-bromophenylalanine (Phe(4-Br)), 2-iodophenylalanine (Phe(2-I)),3-iodophenylalanine (Phe(3-I)), 4-iodophenylalanine (Phe(4-I)),2-trifluoromethylphenylalanine (Phe(2-CF₃)),3-trifluoromethylphenylalanine (Phe(3-CF₃)),4-trifluoromethylphenylalanine (Phe(4-CF₃)), 2-methoxyphenylalanine(Phe(2-OMe)), 3-methoxyphenylalanine (Phe(3-OMe)), 2-nitrophenylalanine(Phe(2-NO₂)), 3-nitrophenylalanine (Phe(3-NO₂)), 4-nitrophenylalanine(Phe(4-NO₂)), 2-cyanophenylalanine (Phe(2-CN)), 3-cyanophenylalanine(Phe(3-CN)), 4-cyanophenylalanine (Phe(4-CN)),3,4-dimethoxyphenylalanine (Phe(3,4-di OMe)), 3,4-difluorophenylalanine(Phe(3,4-di F)), 3,5-difluorophenylalanine (Phe(3,5-di F)),2,4-dichlorophenylalanine (Phe(2,4-di Cl)), 3,4-dichlorophenylalanine(Phe(3,4-di Cl)), 4-benzoylphenylalanine (Bpa), 4-carboxyphenylalanine(Phe(4-COOH)), 4,4′-biphenylalanine (Bip),2,3,4,5,6-pentafluorophenylalanine (Phe(F₅)),3,4,5-trifluorophenylalanine (Phe(F₃)), 4-chlorophenylglycine(Phg(4-Cl)), 2-chlorophenylglycine (Phg(2-Cl)), 3-chlorophenylglycine(Phg(3-Cl)), 4-bromophenylglycine (Phg(4-Br)), 2-bromophenylglycine(Phg(2-Br)), 3-bromophenylglycine (Phg(3-Br)), 4-ethylphenylalanine(Phe(4-Et)), 4-ethoxyphenylalanine (Phe(4-OEt)), 4-butoxyphenylalanine(Phe(4-OBu)), O-methyltyrosine (Tyr(Me)), O-benzyltyrosine (Tyr(Bzl)),3,5-dibromotyrosine (Tyr(diBr)), 3,5-diiodotyrosine (Tyr(diI)),homotyrosine (HoTyr), 3-chlorotyrosine (Tyr(3-Cl)), stereoisomersthereof, and combinations thereof.

Suitable derivatives of lysine (Lys), ornithine (Orn) andα,γ-diaminobutyric acid (Dbu) useful with the present invention include,without limitation, Lys38, Lys27, Lys73, Lys55, Lys28, Lys72, Lys12,Lys123, Lys63, Lys124, Lys82, Lys31, Lys15, Lys125, Lys43, Lys24, Lys5,Lys4, Lys50, Lys81, Orn38, Orn27, Orn73, Orn55, Orn28, Orn72, Orn12,Orn123, Orn63, Orn124, Orn82, Orn31, Orn15, Orn125, Orn43, Orn24, Orn5,Orn4, Orn50, Orn81, Dbu38, Dbu27, Dbu73, Dbu55, Dbu28, Dbu72, Dbu12,Dbu123, Dbu63, Dbu124, Dbu82, Dbu31, Dbu15, Dbu125, Dbu43, Dbu24, Dbu5,Dbu4, Dbu50, Dbu81, stereoisomers thereof, and combinations thereof.Derivatives of Orn and Dbu are similar to the lysine derivatives withcorresponding carboxylic acid attached to the side chain of Orn and Dbu,respectively.

Suitable hydrophilic amino acids useful with the present inventioninclude, without limitation, Val, Leu, Ile, Met, and Phe andstereoisomers thereof.

Suitable hydrophobic amino acids useful with the present inventioninclude, without limitation, the compounds of Table 2:

TABLE 2 Hydrophobic amino acids useful in the present invention. No.X_(AA) Structure 1 Ile

2 Ala

3 Abu

4 Leu

5 Pra

6 Chg

7 Nva

8 Phg

9 Cha

10 Ach

11 Ppca

12 Ana

13 Bpa

14 Val

15 Acpc

16 Thi

17 Nle

18 D-Nal-2

19 Aic

20 D-Phe

21 HoPhe

22 Tyr

23 Tyr(Me)

24 Phe(3-Cl)

25 Tyr(diI)

26 Phe(4-Me)

In some other embodiments, the hydrophobic amino acid is selected fromcompounds of Table 3:

TABLE 3 Hydrophobic amino acids useful in the present invention. No.X_(AA) Structure 1 Nle

2 Leu

3 Pra

4 HLe

5 Cpa

6 Cha

In some other embodiments, the hydrophobic amino acid is independentlyselected from leucine (Leu), a leucine analog, phenylalanine (Phe), aphenylalanine analog, proline (Pro), a proline analog, valine (Val),isoleucine (Ile), glycine (Gly), alanine (Ala), Met, norvaline (Nva),1-aminocyclopropane-1-carboxylic acid (Acpc),1-aminocyclobutane-1-carboxylic acid (Acb), α-cyclohexylglycine (Chg),α-aminoisobutyric acid (Aib), α-aminobutyric acid (Abu),3-(2-thienyl)alanine (2-Thi), 3-(3-thienyl)alanine (3-Thi),3-(3-pyridyl)alanine (3-Pal), 3-(2-naphthyl)alanine (Nal-2),2-amino-2-naphthylacetic acid (Ana), 3,5-dinitrotyrosine (Tyr(3,5-diNO₂)), diethylglycine (Deg),4-amino-4-carboxy-1,1-dioxo-tetrahydrothiopyran (Acdt),1-amino-1-(4-hydroxycyclohexyl)carboxylic acid (Ahch),1-amino-1-(4-ketocyclohexyl)carboxylic acid (Akch),4-amino-4-carboxytetrahydropyran (Actp), 3-nitrotyrosine (Tyr(3-NO₂)),1-amino-1-cyclohexane carboxylic acid (Ach), 2-aminoindane-2-carboxylicacid (Aic), (2S,5R)-5-phenylpyrrolidine-2-carboxylic acid (Ppca),4-thiazoylalanine (Tha), 2-aminooctanoic acid (Aoa), 2-aminoheptanoicacid (Aha), and a stereoisomer thereof. Preferably, the proline analogis hydroxyproline.

Suitable negatively charged amino acids useful with the presentinvention include, without limitation, aspartic acid, glutamic acid,α-aminohexanedioic acid, α-aminooctanedioc acid, homoaspartic acid,γ-carboxy-glutamic acid, 4-carboxyphenylalanine, and a stereoisomerthereof. In other embodiments, the negatively charged amino acid isselected from Aad, Bec and Bmc.

Bisphosphonate drugs useful with the present invention include anysuitable bisphosphonate compound. Exemplary bisphosphonate drugsinclude, but are not limited to, Etidronate (Didronel), Clodronate(Bonefos, Loron), Tiludronate (Skelid), Pamidronate (APD, Aredia),Neridronate, Olpadronate, Alendronate (Fosamax), Ibandronate (Boniva),Risedronate (Actonel) and Zoledronate (Zometa). Additionalbisphosphonates are described below in greater detail. One of skill inthe art will appreciate that other bisphosphonates are useful in thepresent invention.

Salts include, but are not limited, to sulfate, citrate, acetate,oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acidphosphate, phosphonic acid, isonicotinate, lactate, salicylate, citrate,tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate,succinate, maleate, gentisinate, fumarate, gluconate, glucaronate,saccharate, formate, benzoate, glutamate, methanesulfonate,ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate(i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Other saltsinclude, but are not limited to, salts with inorganic bases includingalkali metal salts such as sodium salts, lithium salts, and potassiumsalts; alkaline earth metal salts such as calcium salts, and magnesiumsalts; aluminum salts; and ammonium salts, such as ammonium,trimethyl-ammonium, diethylammonium, andtris-(hydroxymethyl)-methyl-ammonium salts. Other salts with organicbases include salts with diethylamine, diethanolamine, meglumine, andN,N′-dibenzylethylenediamine.

Linkers useful in the present invention includes those possessing one ormore different reactive functional groups that allow for covalentattachment of moieties such as a peptides, monomers, and polymers. Thelinking moiety possesses two or more different reactive functionalgroups. In some cases multivalent linkers can be used and multiplepeptides of the present invention and/or multiple active agents can belinked via the linker. Suitable linkers include, without limitation,those available from Pierce Biotechnology, Inc. (Rockford, Ill.). Oneskilled in the art understands that any reactive functional group can bepresent on the linker, as long as it is compatible with a functionalgroup on the moiety that is to be covalently attached. Some suitablelinkers include, but are not limited to, β-alanine, 2,2′-ethylenedioxybis(ethylamine)monosuccinamide (Ebes), bis(Ebes)-Lys, and polyethyleneglycol. Other suitable linkers include those with biotin. Additionallinkers can be found in Bioconjugate Techniques, Greg T. Hermanson,Academic Press, 2d ed., 2008 (incorporated by reference in its entiretyherein).

A person of ordinary skill in the art will recognize that other linkersare possible with the compounds of the present invention. Many suchlinkers can be found in, or prepared by the techniques recited in,“Bioconjugate Techniques” by Greg T Hermanson, Academic Press, SanDiego, 1996, which is hereby incorporated by reference. Furthermore, aperson of ordinary skill in the art will recognize that other linkerscan be prepared based on Click Chemistry synthetic techniques asdescribed in Kolb, H. C., Finn, M. G., Sharpless, K. B., Angew. Chem.Int'l. Ed. 40 (11): 2004-2021 (2001), which is hereby incorporated byreference. Linkers useful with the present invention include those basedon the EBES and PEG moiety. The linker can include either 0 to 6 EBES orPEG groups. EBES and PEG groups can be conjugated by the techniquesreferenced above.

Acid addition salts, such as mineral acids, organic carboxylic andorganic sulfonic acids, e.g., hydrochloric acid, methanesulfonic acid,maleic acid, are also possible provided a basic group, such as pyridyl,constitutes part of the structure.

The neutral forms of the compounds can be regenerated by contacting thesalt with a base or acid and isolating the parent compound in theconventional manner. The parent form of the compound differs from thevarious salt forms in certain physical properties, such as solubility inpolar solvents, but otherwise the salts are equivalent to the parentform of the compound for the purposes of the present invention.

In some embodiments, the portion of the compound bonded to Z of FormulaI has a formula selected from:

In some other embodiments, the portion of the compound of Formula Ibonded to Z has a formula selected from:

In some embodiments, the portion of the compound of Formula I bonded toZ has a formula selected from:

In some other embodiments, the portion of the compound of Formula Ibonded to Z has a formula selected from:

In some embodiments, the portion of the compound of Formula I bonded toZ has a formula selected from:

In some other embodiments, the portion of the compound of Formula Ibonded to Z has the formula:

In some embodiments, peptide Z has the following structure:-X₁-X₂-X₃-X₄-Y₁-,

wherein X₁ can be a hydrophobic amino acid or derivatives of lysine,ornithine (Orn) and α,γ-diaminobutyric acid (Dbu); X₂ can be anegatively charged amino acid; X₃ can be a hydrophobic amino acid; X₄can be a naturally-occurring amino acid, an unnatural amino acid, or aD-amino acid; Y₁ can be a peptide fragment having m independentlyselected amino acids; and m is an integer of from 0 to 20. In anotherembodiment, m is an integer of from 0 to 15, preferably of from 0 to 10,more preferably of from 0 to 5, and still more preferably of from 0 to3. In another embodiment, Y₁ has a carboxyl-terminal group selected fromthe group consisting of an amide group and a carboxylic acid group.

In some other embodiments, peptide Z has the following structure:-X₁-X₂-X₃-Y₁-,wherein X₁, X₂, X₃ and Y₁ are as defined above.

In other embodiments, peptide Z of Formula I has the formula:-X_(AA1)-X_(AA2)-X_(AA3)-(X_(AA4))_(p)-.X_(AA1) is selected from a hydrophobic amino acid and derivatives oflysine, homolysine (Hly), ornithine (Orn) and α,γ-diaminobutyric acid(Dbu). X_(AA2) is a negatively charged amino acid. X_(AA3) is ahydrophobic amino acid. X_(AA4) is selected from a naturally-occurringamino acid, an unnatural amino acid, and a D-amino acid. Subscript p is0 or 1. Suitable hydrophobic amino acids, derivatives of lysine,homolysine (Hly), ornithine (Orn) and α,γ-diaminobutyric acid (Dbu),negatively charged amino acids, naturally-occurring amino acids,unnatural amino acids, and D-amino acids useful with the presentinvention are described above.

In other embodiments, X_(AA1) can be Leu, a leucine analog, Lys38, or astereoisomer thereof. In another embodiment, X_(AA2) can be Asp, Glu,Aad, or a stereoisomer thereof. In a preferred embodiment, X_(AA2) isAad. In yet another embodiment, X_(AA3) can be Leu, a Leu analog, Phe, aPhe analog, Val, Ile, Ala, Nva, Acpc, Chg, Aib, Abu, Aic, NaI-2, Ana, ora stereoisomer thereof. In still yet another embodiment, X_(AA4) can bea hydrophobic amino acid, a negatively charged amino acid, or astereoisomer thereof. Preferably, the hydrophobic amino acid can be Pro,a Pro analog, or a stereoisomer thereof. Preferably, the Pro analog isHyp.

In another embodiment, peptide Z can be -Nle-Aad-Chg-D-Tyr,-Leu-Aad-Chg-D-Gln-D-Tyr, -Cpa-Asp-Phg-D-Glu-D-Ser, -Leu-Aad-Val-Hyp(SEQ ID NO: 1), -Nle-Aad-Val-D-Thr-Hyp-D-Asn, -Cha-Aad-Nle-D-Gln-D-Asn,-Cpa-Glu-Val-D-Asp-D-Ala, -Hle-Aad-Phe-Chg, (SEQ ID NO: 2),-Nle-Asp-Pra-Gly-Hyp (SEQ ID NO: 3), -Lys38-Aad-Leu-D-Pro,-Cha-Asp-Val-D-Glu-D-Gln, -Cpa-Aad-Ile-D-Asp, -Hle-Aad-Aib-D-3-Pal,-Lys38-Glu-Acpc-Nle-D-Asp-D-Gln, -Nle-Asp-Val-Ach-D-Ala,-Leu-Aad-Ala-Hyp, -Cpa-Asp-Nva-D-Glu, -Leu-Aad-Nva-Hyp-D-Glu,-Hle-Asp-Ile-D-Asp-HoSer-D-Asn, -Cpa-Aad-Aib-D-Thi, -Cpa-Aad-Acpc-Hyp(SEQ ID NO: 4), -Cpa-Aad-Val-D-Tyr-D-Asp, -Nle-Asp-Ala-Aad-Aic (SEQ IDNO: 5), -Cha-Asp-HoPhe-Hyp-D-3-Pal-Nle-Ach, -Nle-Aad-Chg-Hyp-Aad (SEQ IDNO: 6), -Nle-Aad-Chg-Hyp-D-Val-D-Asp-D-Asp,-Cpa-Aad-Chg-Pro-Aad-Phe(3-Cl)-Aad (SEQ ID NO: 7),-Cpa-Aad-Chg-Acp-D-Asp-D-Glu, -Nle-Aad-Chg-Hyp-D-Glu-Ach,-Hle-Aad-Val-Hyp-Chg (SEQ ID NO: 8), -Nle-Glu-Phg-Acp-Aad (SEQ ID NO:9), -Nle-Aad-Val-D-Glu, -Lys38-Aad-Acpc-D-Asp,-Lys38-Aad-Acpc-D-Asn-D-Asn, -Lys38-Aad-D-Phe-D-3-Pal,-Nle-Aad-Cha-D-Glu, -Hle-Aad-Ile-D-Asp-Nle, -Lys38-Aad-Aic-D-Glu-D-Tyr,-Cpa-Aad-Nle-D-Pro, -Lys-Aad-Chg-D-Glu, -Cpa-Aad-Chg-D-Ser-Gly,-Cpa-Aad-Nle-Aad (SEQ ID NO: 10), -Cpa-Aad-Acpc-Aad (SEQ ID NO: 11),-Leu-Aad-Acpc-Aad (SEQ ID NO: 12), -Nle-Aad-Nle-Chg-D-Glu,-HoPhe-Aad-D-Nal-2-D-Glu, -Lys38-Aad-D-Phe-4-Pal-D-Asn,-Lys38-Aad-D-Phe-D-Asp, -Lys38-Aad-D-Phe-D-Ser-Nva, or-Lys38-Aad-D-Phe-D-Val.

In some other embodiments, peptide Z can be-HoPhe-Asp-Phg-Pro-Gly-D-Tyr-Aad, -Hle-Asp-Ile-Pro-Chg (SEQ ID NO: 13),-Cpa-Asp-Ile-Hyp-D-Thr-D-Asn-Nva, -Cha-Asp-Pra-Pro-D-Pro-Gly-D-Ser,-Cha-Asp-Leu-Hyp-HoCit-HoCit (SEQ ID NO: 14), -Lys12-Aad-Nva-Hyp-Hyp(SEQ ID NO: 15), -Hle-Asp-Val-Pro-D-3-Pal-Nva-Ana,-Cpa-Asp-Abu-Acp-Nva-D-Asp, -Cha-Asp-Tyr-Pro-D-His,-Leu-Aad-Abu-Ppca-Ach-D-Tyr, -Leu-Asp-Nva-Hyp-Gly-D-Phe-Nva,-Nle-Asp-Ile-Pro-Aib-D-HoPhe-Tyr(Me), -Cpa-Glu-Tyr-Pro-Chg-Aad-D-Glu,-Hle-Asp-Nva-Pro-D-Glu, -Nle-Asp-Ile-Hyp-Hyp (SEQ ID NO: 16),-Ile-Aad-Ile-Ppca-D-Ile, -HoPhe-Asp-Ala-Pro-Aib-D-Ala,-Hle-Glu-Abu-Hyp-HoCit-HoCit (SEQ ID NO: 17),-Leu-Asp-Leu-Ppca-HoCit-D-Thr-D-Pro, -HoPhe-Asp-Nva-Ppca-D-Ala,-Nle-Asp-Val-Pro-HoCit-Gly (SEQ ID NO:18),-Cpa-Aad-Abu-Pro-D-Ala-D-Tyr-D-Phe(4-Me), -Nle-Glu-Ala-D-Thi,-Cha-Asp-Nle-D-Gln, -Hle-Aad-Ile-D-Asp-D-Phe, -Leu-Asp-D-Phe-Aic,-Cpa-Asp-Leu-D-Thi, -HoPhe-Asp-Abu-D-Asn, -Cha-Aad-Val-Ana-Ahch (SEQ IDNO: 19), -Hle-Asp-Acpc-D-Ala, -Leu-Aad-Ana-D-Pro,-Lys38-Asp-Phe(3-Cl)-D-Pro, -Lys 12-Asp-Nle-Hyp-D-Glu,-Lys38-Aad-D-Nal-2-D-Thr-D-Bpa, -Cpa-Asp-Ala-D-Thi, or-HoPhe-Asp-Ala-Hyp (SEQ ID NO: 20).

In some embodiments, X_(AA1) of peptide Z of Formula I is lysine-A38(Lys38), X_(AA2) of peptide Z of Formula I is α-aminohexanedioic acid(Aad), X_(AA3) of peptide Z of Formula I is a D-amino acid, andsubscript p is 0. In some other embodiments, peptide Z of Formula I isselected from -Lys38-Aad-D-Phe, -Lys38-Aad-Ach, -Lys38-Aad-D-Nal-2,-Lys38-Aad-Ile, -Lys38-Aad-Val, and -Lys38-Aad-Leu. In otherembodiments, the present invention provides compounds of Formula I,wherein peptide Z is -Lys38-Aad-Ach.

In some embodiments, the linker L of Formula I includes at least one ofN-(8-amino-3,6-dioxa-octyl)succinamic acid (EBES) and polyethyleneglycol (PEG).

In some other embodiments, the linker L of Formula I is selected from:

wherein k is from 0 to 6.

In some embodiments, the portion of the compound of Formula I bonded toD of Formula I has the formula:

R⁴ is selected from H, OH and halogen, R⁵ is selected from H and C₁₋₆alkyl, and subscript t is from 1 to 6.

In some other embodiments, the portion of the compound of Formula Ibonded to D has the formula selected from:

In some embodiments, the compounds of Formula I are selected from:

In some embodiments, the salts, hydrates, solvates, prodrug forms,isomers, and metabolites of compounds of Formula I are provided.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are encompassedwithin the scope of the present invention. Certain compounds of thepresent invention may exist in multiple crystalline or amorphous forms.In general, all physical forms are equivalent for the uses contemplatedby the present invention and are intended to be within the scope of thepresent invention.

Certain compounds of the present invention possess asymmetric carbonatoms (optical centers) or double bonds; the enantiomers, racemates,diastereomers, tautomers, geometric isomers, stereoisomeric forms thatmay be defined, in terms of absolute stereochemistry, as (R)- or (S)-or, as (D)- or (L)- for amino acids, and individual isomers areencompassed within the scope of the present invention. The compounds ofthe present invention do not include those which are known in art to betoo unstable to synthesize and/or isolate. The present invention ismeant to include compounds in racemic and optically pure forms.Optically active (R)- and (S)-, or (D)- and (L)-isomers may be preparedusing chiral synthons or chiral reagents, or resolved using conventionaltechniques.

The present invention also provides compounds which are in a prodrugform. Prodrugs of the compounds described herein are those compoundsthat readily undergo chemical changes under physiological conditions toprovide the compounds of the present invention. Additionally, prodrugscan be converted to the compounds of the present invention by chemicalor biochemical methods in an ex vivo environment. For example, prodrugscan be slowly converted to the compounds of the present invention whenplaced in a transdermal patch reservoir with a suitable enzyme orchemical reagent.

The compounds of the invention can be synthesized by a variety ofmethods known to one of skill in the art (see Comprehensive OrganicTransformations by Richard C. Larock, 1989) or by an appropriatecombination of generally well known synthetic methods. Techniques usefulin synthesizing the compounds of the invention are both readily apparentand accessible to those of skill in the relevant art. The discussionbelow is offered to illustrate certain of the diverse methods availablefor use in assembling the compounds of the invention. However, thediscussion is not intended to define the scope of reactions or reactionsequences that are useful in preparing the compounds of the presentinvention. One of skill in the art will appreciate that other methods ofmaking the compounds are useful in the present invention.

One-bead one-compound (OBOC) combinatorial library methods were used toidentify bisaryl urea peptidomimetics, such as LLP2A, as highly potentand selective ligands for the activated form of α₄β₁ integrin (Peng, L.,et al., Nat Chem Biol, 2006, 2(7): p. 381-9). Additionally, LLP2A, whenappropriately radioconjugated (DOTA/⁶⁴Cu or ⁹⁰Y), was shown to exhibitpotential as a diagnostic or therapeutic agent (DeNardo et al., J NuclMed, 2009, 50: p 625-34).

The “one-bead one-compound” (OBOC) combinatorial library method wasfirst reported in 1991 (Lam et al., Nature, 1991, 354: p. 82-4). Inessence, when a “split-mix” synthesis method (Lam et al., id; Houghtenet al., Nature, 1991, 354: p. 84-6; Furka et al., Int. J. PeptideProtein Res., 1991, 37: p. 487-93) is used to generate a combinatoriallibrary, each bead expresses only one chemical entity (Lam et al., id;Lam et al., Chem. Rev., 1997, 97: p. 411-48). Random libraries ofmillions of beads can then be screened in parallel for a specificacceptor molecule (e.g., receptor, antibody, enzyme, virus, whole cell,etc.). Positive beads are physically isolated for structuraldetermination by microsequencing using automatic Edman degradation (Lamet al., Nature, 1991, 354: p. 82-4).

The one-bead-one compound (OBOC) combinatorial library methodsynthesizes millions of compounds such that each bead displays only onecompound. Through the OBOC combinatorial library methods, LLP2A wasidentified as a ligand that specifically binds to the integrin α₄β₁(IC₅₀=2 pM). LLP2A, when conjugated a to near-infrared fluorescent dye,can be used to image α₄β₁-expressing cells with high sensitivity andspecificity and to guide a therapeutic compound to the α₄β₁-expressinglymphomas (Peng, L., et al., Nat Chem Biol, 2006, 2(7): p. 381-9). Theligand is known to direct compounds to the α₄β₁-expressing lymphomas(Peng, L., et al., Nat Chem Biol, 2006, 2(7): p. 381-9; Peng, L., etal., Mol Cancer Ther, 2008, 7(2): p. 432-7 Aina, O. H., et al., MolPharm, 2007. 4(5): p. 631-51; Aina, O. H., et al., Mol Cancer Ther,2005. 4(5): p. 806-13).

The compounds described herein can be used in combination with oneanother, with other active agents known to be useful in treatingosteoporosis or promoting bone growth, or with adjunctive agents thatmay not be effective alone, but may contribute to the efficacy of theactive agent. In some embodiments, co-administration of the compoundsherein with other agents includes administering one active agent within0.5, 1, 2, 4, 6, 8, 10, 12, 16, 20, or 24 hours of a second activeagent. Co-administration includes administering two active agentssimultaneously, approximately simultaneously (e.g., within about 1, 5,10, 15, 20, or 30 minutes of each other), or sequentially in any order.In some embodiments, co-administration can be accomplished byco-formulation, i.e., preparing a single pharmaceutical compositionincluding both active agents. In other embodiments, the active agentscan be formulated separately. In another embodiment, the active and/oradjunctive agents may be linked or conjugated to one another.

Exemplary compounds of the present invention are shown in FIG. 12.

IV. Pharmaceutical Compositions

In another embodiment, the present invention provides a pharmaceuticalcomposition, including a compound of the present invention and apharmaceutically acceptable excipient.

The compounds of the present invention can be prepared and administeredin a wide variety of oral, parenteral and topical dosage forms. Oralpreparations include tablets, pills, powder, dragees, capsules, liquids,lozenges, cachets, gels, syrups, slurries, suspensions, etc., suitablefor ingestion by the patient. The compounds of the present invention canalso be administered by injection, that is, intravenously,intramuscularly, intracutaneously, subcutaneously, intraduodenally, orintraperitoneally. Also, the compounds described herein can beadministered by inhalation, for example, intranasally. Additionally, thecompounds of the present invention can be administered transdermally.The compounds of the present invention can also be administered byintraocular, intravaginal, and intrarectal routes includingsuppositories, insufflation, powders and aerosol formulations (forexamples of steroid inhalants, see Rohatagi, J. Clin. Pharmacol.35:1187-1193, 1995; Tjwa, Ann. Allergy Asthma Immunol. 75:107-111,1995). Accordingly, the present invention also provides pharmaceuticalcompositions including a pharmaceutically acceptable carrier orexcipient and either a compound of the present invention, or apharmaceutically acceptable salt of a compound of the present invention.

For preparing pharmaceutical compositions from the compounds of thepresent invention, pharmaceutically acceptable carriers can be eithersolid or liquid. Solid form preparations include powders, tablets,pills, capsules, cachets, suppositories, and dispersible granules. Asolid carrier can be one or more substances, which may also act asdiluents, flavoring agents, binders, preservatives, tabletdisintegrating agents, or an encapsulating material. Details ontechniques for formulation and administration are well described in thescientific and patent literature, see, e.g., the latest edition ofRemington's Pharmaceutical Sciences, Maack Publishing Co, Easton Pa.(“Remington's”).

In powders, the carrier is a finely divided solid, which is in a mixturewith the finely divided active component. In tablets, the activecomponent is mixed with the carrier having the necessary bindingproperties in suitable proportions and compacted in the shape and sizedesired. The powders and tablets preferably contain from 5% or 10% to70% of the active compound.

The compositions typically include a conventional pharmaceutical carrieror excipient and may additionally include other medicinal agents,carriers, adjuvants, diluents, tissue permeation enhancers,solubilizers, and the like. Preferably, the composition will containabout 0.01% to about 90%, preferably about 0.1% to about 75%, morepreferably about 0.1% to 50%, still more preferably about 0.1% to 10% byweight of a ligand of the present invention or a combination thereof,with the remainder consisting of suitable pharmaceutical carrier and/orexcipients. Appropriate excipients can be tailored to the particularcomposition and route of administration by methods well known in theart, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES, supra.

Suitable solid excipients include, but are not limited to, magnesiumcarbonate; magnesium stearate; calcium phosphate; calcium silicate;talc; pectin; dextran, dextrin, and cyclodextrin inclusion complexes; alow melting wax; cocoa butter; carbohydrates; sugars including, but notlimited to, lactose, dextrose, sucrose, mannitol, or sorbitol; starchesincluding, but not limited to, starch from corn, wheat, rice, potato, orother plants; cellulose such as methyl cellulose,hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; andgums including arabic, tragacanth, and acacia; as well as proteinsincluding, but not limited to, gelatin, collagen; microcrystallinecellulose, water, saline, syrup, ethylcellulose, and polyacrylic acidssuch as Carbopols, e.g., Carbopol 941, Carbopol 980, Carbopol 981, etc.;lubricating agents; mineral oil; wetting agents; emulsifying agents;suspending agents; preserving agents such as methyl-, ethyl-, andpropyl-hydroxy-benzoates (i.e., the parabens); pH adjusting agents suchas inorganic and organic acids and bases; sweetening agents; andflavoring agents; biodegradable polymer beads. If desired,disintegrating or solubilizing agents may be added, such as thecross-linked polyvinyl pyrrolidone, agar, alginic acid, alginates, or asalt thereof, such as sodium alginate.

A pharmaceutically acceptable carrier may include physiologicallyacceptable compounds that act, for example, to stabilize the compoundsof the present invention or modulate their absorption, or otherexcipients as desired. Physiologically acceptable compounds include, forexample, carbohydrates, such as glucose, sucrose or dextrans,antioxidants, such as ascorbic acid or glutathione, chelating agents,low molecular weight proteins or other stabilizers or excipients. Oneskilled in the art would know that the choice of a pharmaceuticallyacceptable carrier, including a physiologically acceptable compound,depends, for example, on the route of administration of the compounds ofthe present invention and on the particular physio-chemicalcharacteristics of the compounds of the present invention.

Generally, such carriers should be nontoxic to recipients at the dosagesand concentrations employed. Ordinarily, the preparation of suchcompositions entails combining the therapeutic agent with buffers,antioxidants such as ascorbic acid, low molecular weight (less thanabout 10 residues) polypeptides, proteins, amino acids, carbohydratesincluding glucose, maltose, sucrose or dextrins, chelating agents suchas EDTA, glutathione and other stabilizers and excipients. Neutralbuffered saline or saline mixed with nonspecific serum albumin areexemplary appropriate diluents.

Dragee cores are provided with suitable coatings such as concentratedsugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound (i.e., dosage). Pharmaceutical preparations of theinvention can also be used orally using, for example, push-fit capsulesmade of gelatin, as well as soft, sealed capsules made of gelatin and acoating such as glycerol or sorbitol. Push-fit capsules can containcompounds of the present invention mixed with a filler or binders suchas lactose or starches, lubricants such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the compounds of thepresent invention may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycol withor without stabilizers.

For preparing suppositories, a low melting wax, such as a mixture offatty acid glycerides or cocoa butter, is first melted and the activecomponent is dispersed homogeneously therein, as by stirring. The moltenhomogeneous mixture is then poured into convenient sized molds, allowedto cool, and thereby to solidify.

Liquid form preparations include solutions, suspensions, and emulsions,for example, water or water/propylene glycol solutions. For parenteralinjection, liquid preparations can be formulated in solution in aqueouspolyethylene glycol solution.

Aqueous solutions suitable for oral use can be prepared by dissolvingthe active component in water and adding suitable colorants, flavors,stabilizers, and thickening agents as desired. Aqueous suspensionssuitable for oral use can be made by dispersing the finely dividedactive component in water with viscous material, such as natural orsynthetic gums, resins, methylcellulose, sodium carboxymethylcellulose,hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gumtragacanth and gum acacia, and dispersing or wetting agents such as anaturally occurring phosphatide (e.g., lecithin), a condensation productof an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate),a condensation product of ethylene oxide with a long chain aliphaticalcohol (e.g., heptadecaethylene oxycetanol), a condensation product ofethylene oxide with a partial ester derived from a fatty acid and ahexitol (e.g., polyoxyethylene sorbitol mono-oleate), or a condensationproduct of ethylene oxide with a partial ester derived from fatty acidand a hexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate).The aqueous suspension can also contain one or more preservatives suchas ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, oneor more flavoring agents and one or more sweetening agents, such assucrose, aspartame or saccharin. Formulations can be adjusted forosmolarity.

Also included are solid form preparations, which are intended to beconverted, shortly before use, to liquid form preparations for oraladministration. Such liquid forms include solutions, suspensions, andemulsions. These preparations may contain, in addition to the activecomponent, colorants, flavors, stabilizers, buffers, artificial andnatural sweeteners, dispersants, thickeners, solubilizing agents, andthe like.

Oil suspensions can be formulated by suspending a compound of thepresent invention in a vegetable oil, such as arachis oil, olive oil,sesame oil or coconut oil, or in a mineral oil such as liquid paraffin;or a mixture of these. The oil suspensions can contain a thickeningagent, such as beeswax, hard paraffin or cetyl alcohol. Sweeteningagents can be added to provide a palatable oral preparation, such asglycerol, sorbitol or sucrose. These formulations can be preserved bythe addition of an antioxidant such as ascorbic acid. As an example ofan injectable oil vehicle, see Minto, J. Pharmacol. Exp. Ther.281:93-102, 1997. The pharmaceutical formulations of the invention canalso be in the form of oil-in-water emulsions. The oily phase can be avegetable oil or a mineral oil, described above, or a mixture of these.Suitable emulsifying agents include naturally-occurring gums, such asgum acacia and gum tragacanth, naturally occurring phosphatides, such assoybean lecithin, esters or partial esters derived from fatty acids andhexitol anhydrides, such as sorbitan mono-oleate, and condensationproducts of these partial esters with ethylene oxide, such aspolyoxyethylene sorbitan mono-oleate. The emulsion can also containsweetening agents and flavoring agents, as in the formulation of syrupsand elixirs. Such formulations can also contain a demulcent, apreservative, or a coloring agent.

V. Administration

Administration of the ligands of the present invention with a suitablepharmaceutical excipient as necessary can be carried out via any of theaccepted modes of administration. Thus, administration can be, forexample, intravenous, topical, subcutaneous, transcutaneous,transdermal, intramuscular, oral, intra joint, parenteral,intra-arteriole, intradermal, intraventricular, intracranial,intraperitoneal, intralesional, intranasal, rectal, vaginal, or byinhalation. Administration may also be directly to the bone surfaceand/or into tissues surrounding the bone.

The compositions containing a ligand or a combination of ligands of thepresent invention may be administered repeatedly, e.g., at least 2, 3,4, 5, 6, 7, 8, or more times, or the composition may be administered bycontinuous infusion. Suitable sites of administration include, but arenot limited to, skin, bronchial, gastrointestinal, anal, vaginal, eye,and ear. The formulations may take the form of solid, semi-solid,lyophilized powder, or liquid dosage forms, such as, for example,tablets, pills, capsules, powders, solutions, suspensions, emulsions,suppositories, retention enemas, creams, ointments, lotions, gels,aerosols, or the like, preferably in unit dosage forms suitable forsimple administration of precise dosages.

The pharmaceutical preparations are typically delivered to a mammal,including humans and non-human mammals. Non-human mammals treated usingthe present methods include domesticated animals (i.e., canine, feline,murine, rodentia, and lagomorpha) and agricultural animals (bovine,equine, ovine, porcine).

The pharmaceutical preparation is preferably in unit dosage form. Theterm “unit dosage form” refers to physically discrete units suitable asunitary dosages for human subjects and other mammals (e.g., dogs), eachunit containing a predetermined quantity of active material calculatedto produce the desired onset, tolerability, and/or therapeutic effects,in association with a suitable pharmaceutical excipient (e.g., anampoule). In addition, more concentrated compositions may be prepared,from which the more dilute unit dosage compositions may then beproduced. The more concentrated compositions thus will containsubstantially more than, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more times the amount of a ligand or a combination of ligands. Insuch form the preparation is subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofpreparation, such as packeted tablets, capsules, and powders in vials orampoules. Also, the unit dosage form can be a capsule, tablet, cachet,or lozenge itself, or it can be the appropriate number of any of thesein packaged form. The composition can, if desired, also contain othercompatible therapeutic agents. Preferred pharmaceutical preparations candeliver the compounds of the invention in a sustained releaseformulation.

Methods for preparing such dosage forms are known to those skilled inthe art (see, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, 18THED., Mack Publishing Co., Easton, Pa. (1990)). The composition to beadministered contains a quantity of the ligand or combination of ligandsin a pharmaceutically effective amount for relief of a condition beingtreated (e.g. osteoporosis) when administered in accordance with theteachings of this invention. In addition, pharmaceutically acceptablesalts of the ligands of the present invention (e.g., acid additionsalts) may be prepared and included in the compositions using standardprocedures known to those skilled in the art of synthetic organicchemistry and described, e.g., by J. March, Advanced Organic Chemistry:Reactions, Mechanisms and Structure, 4^(th) Ed. (New York:Wiley-Interscience, 1992).

For oral administration, the compositions can be in the form of tablets,capsules, emulsions, suspensions, solutions, syrups, sprays, lozenges,powders, and sustained-release formulations. Suitable excipients fororal administration include pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharine, talcum, cellulose,glucose, gelatin, sucrose, magnesium carbonate, and the like.

In some embodiments, the pharmaceutical compositions take the form of apill, tablet, or capsule, and thus, the composition can contain, alongwith the ligands or combination of ligands, any of the following: adiluent such as lactose, sucrose, dicalcium phosphate, and the like; adisintegrant such as starch or derivatives thereof; a lubricant such asmagnesium stearate and the like; and a binder such a starch, gum acacia,polyvinylpyrrolidone, gelatin, cellulose and derivatives thereof. Theligands can also be formulated into a suppository disposed, for example,in a polyethylene glycol (PEG) carrier.

Liquid compositions can be prepared by dissolving or dispersing a ligandor a combination of ligands and optionally one or more pharmaceuticallyacceptable adjuvants in a carrier such as, for example, aqueous saline(e.g., 0.9% w/v sodium chloride), aqueous dextrose, glycerol, ethanol,and the like, to form a solution or suspension, e.g., for oral, topical,or intravenous administration. The ligands of the present invention canalso be formulated into a retention enema.

For topical administration, the compositions of the present inventioncan be in the form of emulsions, lotions, gels, creams, jellies,solutions, suspensions, ointments, and transdermal patches. For deliveryby inhalation, the composition can be delivered as a dry powder or inliquid form via a nebulizer. For parenteral administration, thecompositions can be in the form of sterile injectable solutions andsterile packaged powders. Preferably, injectable solutions areformulated at a pH of about 4.5 to about 7.5.

The compositions of the present invention can also be provided in alyophilized form. Such compositions may include a buffer, e.g.,bicarbonate, for reconstitution prior to administration, or the buffermay be included in the lyophilized composition for reconstitution with,e.g., water. The lyophilized composition may further comprise a suitablevasoconstrictor, e.g., epinephrine. The lyophilized composition can beprovided in a syringe, optionally packaged in combination with thebuffer for reconstitution, such that the reconstituted composition canbe immediately administered to a patient.

Generally, administered dosages will be effective to deliver picomolarto micromolar concentrations of the ligand to the appropriate site orsites. However, one of ordinary skill in the art understands that thedose administered will vary depending on a number of factors, including,but not limited to, the particular ligand or set of ligands to beadministered, the mode of administration, the type of application (e.g.,imaging, therapeutic), the age of the patient, and the physicalcondition of the patient. Preferably, the smallest dose andconcentration required to produce the desired result should be used.Dosage should be appropriately adjusted for children, the elderly,debilitated patients, and patients with cardiac and/or liver disease.Further guidance can be obtained from studies known in the art usingexperimental animal models for evaluating dosage. However, the increasedcell binding affinity and specificity associated with the ligands of thepresent invention permits a wider margin of safety for dosageconcentrations and for repeated dosing.

Individuals to be treated using methods of the present invention can beany mammal. For example, local increase in bone can be used for fracturehealing, fusion (arthrodesis), orthopedic reconstruction, andperiodontal repair. Systemic increase in bone would be for treatment oflow bone mass, i.e. osteoporosis. Such individuals include a dog, cat,horse, cow, or goat, particularly a commercially important animal or adomesticated animal, more particularly a human.

In other embodiments, the present invention provides a method ofpromoting systemic bone growth. Systemic bone growth refers to thegrowth of bone throughout the subject, and can effect all the bones inthe subject's body. A subject in need of systemic bone growth can sufferfrom a variety of ailments and disease states. In some embodiments, thesubject suffers from a low bone mass phenotype disease. Low bone masscan be determined by a variety of methods known to one of skill in theart. For example, low bone mass can be characterized by a T-score lessthan about −1. Low bone mass phenotype diseases can includeosteoporosis, osteopenia, and osteoporosis-pseudoglioma syndrome (OPPG).In some other embodiments, the low bone mass phenotype disease can beosteopenia or osteoporosis-pseudoglioma syndrome (OPPG).

Following administration of the compounds of the present invention,systemic bone growth can be determined by a variety of methods, such asimprovements in bone density. Bone density can be measured by a varietyof different methods, including the T-score and Z-score. The T-score isthe number of standard deviations above or below the mean for a healthy30 year old adult of the same sex as the patient. Low bone mass ischaracterized by a T-score of −1 to −2.15. Osteoporosis is characterizedby a T-score less than −2.15. The Z-score is the number of standarddeviations above or below the mean for the patient's age and sex.Improvement in the T-score or Z-score indicate bone growth. Bone densitycan be measured in a variety of places of the skeleton, such the spineor the hip. One of skill in the art will appreciate that other methodsof determining bone density are useful in the present invention.

The pharmaceutical compositions of the present invention can be preparedfor administration by a variety of different routes. In general, thetype of carrier is selected based on the mode of administration.Pharmaceutical compositions can be formulated for any appropriate mannerof administration, including, for example, topical, oral, nasal,intrathecal, rectal, vaginal, sublingual or parenteral administration,including subcutaneous, intravenous, intramuscular, intrasternal,intracavernous, intrameatal, or intraurethral injection or infusion. Apharmaceutical composition (e.g., for oral administration or delivery byinjection) can be in the form of a liquid (e.g., an elixir, syrup,solution, emulsion or suspension). A liquid pharmaceutical compositionmay include, for example, one or more of the following: sterile diluentssuch as water for injection, saline solution, preferably physiologicalsaline, Ringer's solution, isotonic sodium chloride, fixed oils that mayserve as the solvent or suspending medium, polyethylene glycols,glycerin, propylene glycol or other solvents; antibacterial agents;antioxidants; chelating agents; buffers such as acetates, citrates orphosphates and agents for the adjustment of tonicity such as sodiumchloride or dextrose. A parenteral preparation can be enclosed inampoules, disposable syringes or multiple dose vials made of glass orplastic. The use of physiological saline is preferred, and an injectablepharmaceutical composition is preferably sterile.

The formulations of the invention are also suitable for administrationin all body spaces/cavities, including but not limited to pleura,peritoneum, cranium, mediastinum, pericardium, bursae or bursal,epidural, intrathecal, intraocular, intra-articular, intra-discal,intra-medullary, perispinal, etc.

Some slow release embodiments include polymeric substances that arebiodegradable and/or dissolve slowly. Such polymeric substances includepolyvinylpyrrolidone, low- and medium-molecular-weight hydroxypropylcellulose and hydroxypropyl methylcellulose, cross-linked sodiumcarboxymethylcellulose, carboxymethyl starch, potassiummethacrylatedivinylbenzene copolymer, polyvinyl alcohols, starches,starch derivatives, microcrystalline cellulose, ethylcellulose,methylcellulose, and cellulose derivatives, β-cyclodextrin, poly(methylvinyl ethers/maleic anhydride), glucans, scierozlucans, mannans,xanthans, alzinic acid and derivatives thereof, dextrin derivatives,glyceryl monostearate, semisynthetic glycerides, glycerylpalmitostearate, glyceryl behenate, polyvinylpyrrolidone, gelatine,agnesium stearate, stearic acid, sodium stearate, talc, sodium benzoate,boric acid, and colloidal silica.

Slow release agents of the invention may also include adjuvants such asstarch, pregelled starch, calcium phosphate mannitol, lactose,saccharose, glucose, sorbitol, microcrystalline cellulose, gelatin,polyvinylpyrrolidone, methylcellulose, starch solution, ethylcellulose,arabic gum, tragacanth gum, magnesium stearate, stearic acid, colloidalsilica, glyceryl monostearate, hydrogenated castor oil, waxes, andmono-, bi-, and trisubstituted glycerides. Slow release agents may alsobe prepared as generally described in WO94/06416.

In practicing the methods of the present invention, the pharmaceuticalcompositions can be used alone, or in combination with other therapeuticor diagnostic agents. The additional drugs used in the combinationprotocols of the present invention can be administered separately or oneor more of the drugs used in the combination protocols can beadministered together, such as in an admixture. Where one or more drugsare administered separately, the timing and schedule of administrationof each drug can vary. The other therapeutic or diagnostic agents can beadministered at the same time as the compounds of the present invention,separately or at different times.

VI. Methods of Treating

In some embodiments, the present invention provides a method of treatingosteoporosis, wherein the method includes administering to a subject inneed thereof, a therapeutically effective amount of a compound ofFormula I.

The present invention also provides methods of treating diseasescharacterized by secondary induced osteoporosis (low bone mass)including, but not limited to, osteomalacia, polyostotic fibrousdysplasia, Paget's disease, rheumatoid arthritis, zero gravity,osteoarthritis, prolonged inactivity or immobility, osteomyelitis,celiac disease, Crohn's disease, ulcerative colitis, inflammatory bowldisease, gastrectomy, secondary induced osteoporosis, amennorhea,Cushing's disease, Cushing's syndrome, diabetes mellitus, diabetes,eating disorders, hyperparathyroidism, hyperthyroidism,hyperprolactinemia, Kleinefelter syndrome, thyroid disease, Turnersyndrome, steroid induced osteoporosis, seizure or depression inducedosteoporosis, immobility, arthritis, cancer induced secondaryosteoporosis, gonadotropin-releasing hormone agonists induced low bonemass, thyroid medication induced low bone mass, dilantin (phenyloin),depakote induced low bone mass, chemotherapy induced low bone mass,immunosuppressant induced low bone mass, blood thinning agents inducedlow bone mass, Grave's disease, juvenile rheumatoid arthritis,malabsorption syndromes, anorexia nervosa, kidney disease,anticonvulsant treatment (e.g., for epilepsy), corticosteroid treatment(e.g., for rheumatoid arthritis, asthma), Immunosuppressive treatment(e.g., for cancer), inadequate nutrition (especially calcium, vitaminD), excessive exercise leading to amenorrhea (absence of periods),smoking, and alcohol abuse, pregnancy-associated osteoporosis, copperdeficiency, dibasic aminoaciduria type 2, Werner's syndrome,Hajdu-Cheney syndrome, hyperostosis corticalis deformans juvenilis,methylmalonic aciduria type 2, cystathionine beta-synthase deficiency,exemestane, hyperimmunoglobulin E (IgE) syndrome, Haemochromatosis,Singleton-Merten syndrome, beta thalassaemia (homozygous), reflexsympathetic osteodystrophy, sarcoidosis, Winchester syndrome,Hallermann-Streiff syndrome (HSS), cyproterone, glycerol kinasedeficiency, Bonnet-Dechaume-Blanc syndrome, prednisolone, heparin,geroderma osteodysplastica, Torg osteolysis syndrome, orchidectomy,Fabry's disease, pseudoprogeria syndrome, Wolcott-Rallison syndrome,ankylosing spondylitis, myeloma, systemic infantile hyalinosis,Albright's hereditary osteodystrophy, autoimmune lymphoproliferativesyndrome, Brown-Sequard syndrome, Diamond-Blackfan anemia,galactorrhoea-hyperprolactinaemia, gonadal dysgenesis, kidneyconditions, Menkes disease, menopause, neuritis, ovarian insufficiencydue to FSH resistance, familial ovarian insufficiency, premature aging,primary biliary cirrhosis, prolactinoma, familial prolactinoma, renalosteodystrophy, ulcerative colitis, underweight, Werner syndrome, bonetumor, bone cancer, brittle bone disease, osteogenesis imperfectacongenita, and osteogenesis imperfecta tarda. Other conditions include abone injury, such as a fracture or weakened bone, or bone injured due toradiation treatment. One of skill in the art will appreciate that othertypes of conditions, diseases and treatments lead to osteoporosis.

The present invention also provides methods of treating patientpopulations characterized by injured bone, such as fractured bone orbone injured due to radiation, as well as children for whom osteoporosismedications are contraindicated.

In some embodiments, the present invention provides a method ofpromoting bone growth by administering to a subject in need thereof atherapeutically effective amount of a compound of the present invention.

Bone growth can be measured in a variety of ways known to one of skillin the art. Methods of measuring bone growth include, but are notlimited to, Uct (micro CT), Dual X-ray absorption (Bone density),ultrasound, QCT, SPA, DPA, DXR, SEXA, QUS, X-ray, using the human eyeduring surgically manipulation, Alizarin red S, serum osteocalcin, serumalkaline phosphatase, Serum bone Gla-protein (BGP), bone mineralcontent, serum calcium, serum phosphorus, tantalum markers, and serumIGF-1.

Many indicators of bone growth can be used to measure bone growth,including bone density. In some embodiments, bone growth can bedemonstrated by an increase of 0.1% in bone density. In otherembodiments, bone growth can be demonstrated by an increase of 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 5, 10, 20, 30, 40, 50, 60, 70, 80,90, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000% or greater, inbone density.

One of skill in the art appreciates that bone growth be local, systemicor both.

In some other embodiments, the method of the present invention promotesbone growth by administering the compound of the present invention, suchas a compound of formula I. Administration of a compound of the presentinvention can promote local bone growth and/or systemic bone growth. Insome embodiments, the administration of a compound of the presentinvention promotes systemic bone growth. Bone growth can be achieved byincreasing bone mineral content, increasing bone density and/or growthof new bone. In other embodiments, local application of the compound ofthe present invention and a drug achieves systemic bone growth.

In some other embodiments, the present invention provides a method oftreating low bone mass by administering to a subject in need thereof atherapeutically effective amount of a compound of the present invention.

VII. Examples Example 1 Synthesis of LLP2A-Alendronate (LLP2A-Ale)

LLP2A-Alendronate (LLP2A-Ale) is made by conjugate addition of thesulfhydryl group of LLP2A-Lys(D-Cys) to Alendronate-maleimide (Ale-Mal),the latter being prepared in situ from alendronate andsulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate(sulfo-SMCC). LLP2A-Lys(D-Cys) is prepared by solid phase synthesis fromseveral commercially available starting materials and one characterizedintermediate, 4-[(N′-2-methylphenyl)ureido]phenylacetic acid (UPA),which was also prepared from commercially available starting materials.

Synthesis of 4-[(N′-2-methylphenyl)ureido]phenylacetic acid (UPA)

4-[(N′-2-methylphenyl)ureido]phenylacetic acid (UPA) was synthesized asoutlined in FIG. 10A, Synthesis of UPA. In brief, UPA was synthesized byadding o-Tolyl isocyanate (19.646 mL, 158.47 mmol) dropwise to asuspension of 4-aminophenylacetic acid (23.8 g, 156.9 mmol) inN,N-dimethylformamide (DMF) (62 mL) with stirring. The mixture graduallybecame clear and was allowed to stir for 2 hours. The resulting solutionwas poured into ethyl acetate (700 mL) with stirring. The off-whiteprecipitate was filtered and washed with ethyl acetate (3×100 mL) andacetonitrile (ACN) (3×100 mL), respectively. The solid was dried invacuo. Weight (off-white powder): 36.7 g (82.3% crude yield). Orbitraphigh resolution Electrospray Ionization Mass Spectrometry (ESI-MS)[M+H]⁺: 285.1197 (calculated: 285.1239), [M+Na]⁺: 307.1010 (calculated:307.1059), [M+K]⁺: 323.0746 (calculated: 323.0798).

Solid Phase Synthesis of LLP2A-Lys(D-Cys)

LLP2A-Lys(D-Cys) was synthesized as outlined in FIG. 10B, Solid phasesynthesis of LLP2A-Lys(D-Cys).

Rink amide MBHA resin (0.5 g, 0.325 mmol, loading 0.65 mmol/g) wasswollen in DMF for 3 hours before Fmoc-deprotection with 20%4-methylpiperidine in DMF twice (5 and 15 minutes, respectively). Thebeads were then washed with DMF (3×10 mL), methanol (MeOH) (3×10 mL) andDMF (3×10 mL), respectively. Fmoc-Lys(Dde)-OH (0.519 g, 0.975 mmol) wasdissolved in a solution of N-Hydroxybenzotriazole (HOBt) (0.149 g, 0.975mmol) and N,N′-diisopropylcarbidiimide (DIC) (152 uL, 0.975 mmol) in DMF(8 mL), which was then added to the suspension of the beads. Thecoupling was carried out at room temperature overnight. Afterfiltration, the beads were washed with DMF (3×10 mL), MeOH (3×10 mL),and DMF (3×10 mL), respectively.

After removal of Fmoc, the beads were then subjected to two cycles ofcoupling with and deprotection of the Fmoc-linker in the same manner asdescribed above. The beads were washed with DMF (3×10 mL), MeOH (3×10mL) and DMF (3×10 mL). Fmoc-Ach-OH (0.365 g, 0.975 mmol) was dissolvedin a solution of HOBt (0.149 g, 0.975 mmol) and DIC (152 uL, 0.975 mmol)in DMF, and was then added into the beads. The coupling was carried outat room temperature for 2 hours. After filtration, the beads were washedwith DMF (3×10 mL), MeOH (3×10 mL), and DMF (3×10 mL), respectively. TheFmoc deprotection group was removed with 20% 4-methylpiperidine twice((5 and 15 minutes, respectively)).

After washing with DMF, MeOH, and DMF respectively, the beads were thensubjected to additional coupling and deprotection cycles stepwise withFmoc-Aad(OtBu)-OH and Fmoc-Lys(Alloc)-OH in the same manner as describedabove. After removal of Fmoc, a solution of UPA (0.923 g, 3.25 mmol),HOBt (0.498 g, 3.25 mmol) and DIC (509 μL, 3.25 mmol) in DMF was addedto the beads. The reaction was conducted at room temperature untilKaiser test negative (3 hours to overnight). The beads were washed withDMF (3×10 mL), methanol (3×10 mL), and DMF (3×10 mL). The Allocprotecting group was removed by treating with Pd(PPh₃)₄ (0.2 eq.) andPhSiH₃ (20 eq.) in dichloromethane (DCM), twice (30 minutes, each).

A solution of trans-3-(3-pyridyl)acrylic acid (0.37 g, 1.3 mmol), HOBt(0.176 g, 1.3 mmol) and DIC (201 μL, 1.3 mmol) in DMF (8 mL) was addedto the beads. The coupling proceeded at room temperature 4 hours toovernight until Kaiser test was negative. The beads were washed with DMF(5×5 mL), MeOH (3×5 mL) and DCM (3×5 mL). The Dde protecting group wasremoved with 2% NH₂NH₂ in DMF twice (5 and 10 minutes, respectively).The beads were washed with DMF, MeOH and DMF, followed by coupling of (4eq. to resin, 220 mg, 1.3 mmol) Boc-D-Cys(Trt)-OH, HOBt (0.176 g, 1.3mmol) and DIC (201 μL, 1.3 mmol) in DMF (8 mL). The coupling reactionwas conducted at room temperature until Kaiser test negative (4 hours toovernight). The beads were thoroughly washed with DMF, MeOH and DCM,respectively, and then dried under vacuum for 1 hour before adding acleavage mixture of 82.5% trifluoroacetic acid (TFA): 5% thioanisole: 5%phenol: 5% water: 2.5% triisopropylsilane (TIS) (v/v). The cleavagereaction was conducted at room temperature over 2-3 hours. The off-whitecrude product was precipitated out and washed with cold ether. Thepurity was determined by analytical reverse phase high performanceliquid chromatography (RP-HPLC) and the crude product was used in thenext step without further purification. LLP2A-Lys(D-Cys) MALDI-TOF MS[M+H]⁺: 1502.88 (calculated: 1502.77); [M+Na]⁺: 1524.88 (calculated:1524.75).

Synthesis of LLP2A-Ale

LLP2A-Ale was synthesized through conjugate addition of LLP2A-Lys(D-Cys)to Ale-Mal, formed in situ from alendronate and sulfo-SMCC. Thesynthetic scheme is shown in FIG. 10C, Preparation of LLP2A-Ale throughconjugating LLP2A-Lys(D-Cys) with Ale-Mal.

Alendronate disodium salt (1.0 eq.) (powder from lyophilization ofaqueous solution of alendronic acid and 2 eq. NaOH) was dissolved in 0.1M phosphate buffered saline (PBS) (with 10 mM ethylenediaminetetraacetic acid), pH7.5. The aqueous solution was then cooled in an icewater bath, and a solution of Sulfo-SMCC (1.1 eq.) in water was addeddropwise. Following completion of addition, the resulting solution wasallowed to warm to room temperature while being stirred for 2 hours.This solution was cooled before the dropwise addition of a solution ofLLP2A-Lys(D-Cys) (1.0 eq.) in a small amount of 50% acetonitrile/water.The pH was adjusted to between 6 and 7 with aq. NaHCO₃, if needed. Theresulting mixture was allowed to warm to room temperature and stirredfor 1 hour or until Ellman test negative, and then lyophilized. Theresulting powder was redissolved in a small amount of 50% ACN/water andpurified by RP-HPLC (C18 column). Buffer A: 0.5% acetic acid/H₂O. BufferB: 0.5% acetic acid/ACN. The collected eluent was lyophilized to give awhite powder. The identity of LLP2A-Ale was confirmed withMatrix-assisted laser desorption/ionization time of flight massspectrometry (MALDI-TOF MS)[M+H]⁺: 1970.78 (calculated: 1970.88).

Example 2 Synthesis of LLP2A-Alendronate (1) [LLP2A-Ale (1)]

LLP2A-Ale (1) was synthesized through conjugation of Alendronate-SH(Ale-SH) to LLP2A-Mal via Michael addition. The synthetic scheme isshown in FIG. 11.

Preparation 1a: Synthesis of Ale-SH.

The synthesis of Ale-SH was achieved by reacting alendronate with 2-IT(1 equivalent) in PBS at pH 7.5 for 1 hour followed by precipitationwith EtOH. The solid was dissolved in water and precipitated with EtOH,twice.

Preparation 1b: Synthesis of LLP2A-Mal.

LLP2A-Mal was prepared with maleimide attached to the side chain of Lysand two hydrophilic linkers between LLP2A and Lys(Mal) using similarapproach described above. The synthesis was performed on rink amide MBHAresin by standard solid-phase peptide synthesis approach using Fmoc/tBuchemistry and HOBt/DIC coupling. The synthetic scheme is shown in FIG.11. Fmoc-Lys(Dde)-OH was first coupled to the resin, followed bycoupling of two linkers. LLP2A was then prepared as previously reportedon the N-terminus of the linker using similar approach described above.The Dde protecting group was removed with 2% NH₂NH₂ in DMF twice (5 min,10 min). The beads were washed with DMF, MeOH and DMF, followed bycoupling of (3 equivalents to resin) 3-maleimido propionic acid andHOBt, DIC. The coupling reaction was conducted at room temperatureovernight. The beads were thoroughly washed with DMF, MeOH and DCM andthen dried under vacuum for 1 hour before adding a cleavage mixture of95% TFA: 2.5% water: 2.5% TIS. Cleavage of compounds from the resin andremoval of protecting group were achieved simultaneously over 2 hours atroom temperature. The off-white crude products were precipitated withcold ether and purified by semi-preparative reversed-phase highperformance liquid chromatography (RP-HPLC) to give LLP2A-Mal. Theidentity of LLP2A-Mal was confirmed by MALDI-TOF MS [M+Na]⁺: 1572.76(calculated: 1549.78).

Preparation 1c: Synthesis of LLP2A-Ale (1).

LLP2A-Ale (1) was prepared by conjugating LLP2A-Mal with Ale-SH. Ale-SHwas dissolved in PBS buffer at pH 7.2 containing 5% DMSO. Subsequently,a solution of LLP2A-Mal (1.2 equivalents) in a small amount of DMSO wasadded to the Ale-SH solution. The resulting mixture was stirred at roomtemperature for 1 hour and then lyophilized. The powder was re-dissolvedin a small amount of water and passed through Varian MEGA BOND ELUT C18column (60 mL, 40 um particle size) and eluted with water, 5% ACN/water,10% ACN/water and 50% ACN/water. The eluents were collected and checkedby mass spectroscopy. The eluents with pure product were combined andlyophilized to give white powder. MALDI-TOF MS [M+H]⁺: 1900.78(calculated: 1899.83).

Example 3 Osteogenic Differentiation of Bone Marrow Stem Cells with aHigh Affinity to LLP2A

A new color-encoding method that facilitates high-throughput screeningof OBOC combinatorial libraries was developed. This method differs fromthe traditional methods used to monitor integrin expressions in theMSCs, i.e., the use of antibody-based fluorescence-activated cellsorting (FACS) or immunoblotting. In the present method, polymer beadsdisplaying chemical compounds or families of compounds were stained withoil-based organic dyes that are used as coding tags. The color dyes didnot affect cell binding to the compounds displayed on the surface of thebeads. These rainbow beads were prepared in a multiplex manner such thateach ligand reacted to one or a series of integrins that were coded onecolor, See FIG. 7 (Luo, J., et al., J Comb Chem, 2008, 10(4): p.599-604). This rainbow bead technique was then applied to determine theintegrin profile on the cell surfaces. By incubating the bone marrowcells undergoing osteogenic differentiation with the rainbow beads, wefound that α₄β₁ integrin was highly expressed in these osteoprogenitorcells and had high affinity to LLP2A as shown in FIG. 7.

In FIG. 7, bone marrow cells were cultured in osteogenic medium for 7days and incubated with rainbow beads. The purple beads that coded withLLP2A, the ligand with high affinity and specificity against α4β1, werecovered with layers of cells identified by green arrows. Originalmagnification, 10×, is shown on the right side of FIG. 7, and 20×magnification is shown on the left side of FIG. 7. Weak bindings werealso seen in the black, purple and green beads, coded RGD1, RGD2 andLYK1 ligands respectively. All of these beads had non-specific bindingsto many other integrins.

Example 4 Human-MSCs Directed to Bone with LLP2A-Ale for Promoting BoneGrowth

Human MSCs (huMSCs) were obtained and injected together with LLP2A-Aleto immunodeficient mice, NOD/SCID/mucopolysaccharidosis type VII(MPSVII). This mouse strain lacked the β-glucuronidase (GUSB) enzyme.The donor cells could be easily detected using biochemical detection ofβ-glucuronidase (See Meyerrose, T. E., et al., Stem Cells, 2007. 25(1):p. 220-7; Meyerrose, T. E., et al., Stem Cells, 2008. 26(7): p.1713-22). At three-month of age, the mice received a single intravenous(i.v.) injection of either vehicle (100 μl PBS/mouse), huMSCs (5×10⁵/100μl/PBS/mouse), huMSCs+LLP2A (5 nM/mouse) or huMSCs+LLP2A-Ale (500ng/mouse; total compound weight 3.5 μg, with alendronate concentrationof 500 ng/mouse). The alendronate dose we used for the study wasapproximately one-fifth of the therapeutic dose for the treatment ofosteoporosis (See Colon-Emeric, C. S., JAMA, 2006, 296(24): p. 2968-9;Ma, Y. L., et al., Endocrinology, 2003. 144(5): p. 2008-15). As shown inFIG. 8, LLP2A-Ale greatly increased the number of huMSCs on the bonesurface of huMSCs to bone as compared to the control groups (PBS andhuMSCs) 24 hours after the huMSCs injection. As shown in FIG. 1, threeweeks after the transplantation, the huMSCs were only seen adjacent tothe bone surface in LLP2A-Ale treated group. HuMSC, as shown by GUSB+redstain, were only present at the trabecular bone surface in theLLP2A-Ale-treated group (lower panel). There was no demonstrable GUSBactivity in the lung, liver, kidney and LVB that received PBS (toppanels) or huMSCs (middle panel). Cancellous bone mass in the vertebralbody was—more than 40% higher in the group treated with MSC+LLP2A-Ale(cancellous bone volume, BV/TV 13.9±2.2%) compared to PBS (BV/TV9.4±1.7%), MSC (BV/TV 10.2±1.9%) or MSC+LLP2A (BV/TV 10.5±0.5%) controlsthree weeks after a single injection of huMSC and LLP2A-Ale. Thisincrease in cancellous bone mass was accompanied by significantincreases in biochemical bone formation parameters such as boneformation markers, procollagen 1 N-terminal propeptide (P1NP), andosteocalcin, see FIG. 2 a, and surfaced-based bone formation parameterssuch as osteoblast surface, mineral apposition rate or surface-basedbone formation rate (BFR/BS), See FIG. 2 b-c. Osteoclastic resorptiondid not differ between the groups (CTX-1), See FIG. 2 a. As shown inFIG. 3, bone formation rates at both the endocortical and periostealsurfaces of the mid-femur also increased by 50-100%, with markedlyincreased in intra-cortical bone remodeling in huMSC+LLP2A-Ale treatedgroup. These preliminary data demonstrate that LLP2A-Ale can direct MSCsto bone to enhance both cancellous and cortical bone formation and bonemass.

In FIG. 8, Three-month-old MSPVII mice received a single intravenousinjection of either of PBS, huMSC (5×10⁵) or huMSC+LLP2A or LLP2A-Ale.The mice were sacrificed 24 hours later. Following sacrifice, smallportions of organ including lumbar vertebral bodies (LVB) were harvestedand frozen in Optimal Cutting Temperature embedding media. The sectionswere stained using naphthol-AS-BI-β-Dglucuronide (GUSB) as a substrate.Representative sections from lung, liver, kidney and lumbar vertebral(LVB) trabecular bone regions were presented. Human MSCs, as showed byGUSB+red stains, were seen accumulated in lung, liver and kidney at24-hours in huMSC, huMSC+LLP2A or huMSC+LLP2A-Ale groups. Significantamount of these cells were also seen adjacent to bone surface only inhuMSCs+LLP2A-Ale group (lower panels). Isolated GUSB-positive cells werealso observed within bone marrow of the LVB in MSC (middle panel) group.

NOD-SCID MPSVII Mice.

The NOD-SCID MPSVII strain was the result of extensive backcrossing ofthe mutant GUSB allele from the B6.C-H-2^(bm1)/ByBirgus^(mps/+) mouseonto the NOD/LtSz-scid background. Animals were bred and maintained atthe Stem Cell Department of the UC Davis Medical Center under approvedanimal care protocols. Affected animals were generated by breeding miceheterozygous for the MPSVII mutation. Homozygous GUSB-deficient pupswere identified at birth by biochemical analysis of toe tissue.

Biochemical Markers of Bone Turnover.

Serum levels of the amino terminal propeptide of type 1 procollagen(P1NP), osteocalcin and C-terminal telopeptides of type I collagen(CTX-I) were measured using mouse sandwich ELISA kits from BiomedicalTechnologies (Stroughton, Mass.) or Immunodiagnostic System (FountainHills, Ariz.). The manufacturer's protocols were followed and allsamples were assayed in duplicate. A standard curve was generated fromeach kit and the absolute concentrations were extrapolated from thestandard curve. The coefficients of variations (CVs) for inter-assay andintra-assay measurements were less than 10% for all assays and aresimilar to the manufacturer's references (Yao, W., et al., ArthritisRheum, 2008, 58(6): p. 1674-86; Yao, W., et al., Arthritis Rheum, 2008,58(11): p. 3485-3497).

Example 5 LLP2A-Ale Augmented Bone Formation and Bone Mass

Two-month-old female 129SvJ mice received intravenous (i.v.) injectionsof vehicle (PBS), 2A (5 nM), Ale (250 ng/mouse) or LLP2A-Ale[alendronate concentration of 10 ng/mouse (70 ng total compound weight)or alendronate concentration of 250 ng/mouse (1.75 μg total compoundweight)]. The dose of alendronate used for the experiments were at leastone-tenth lower than of the therapeutic dose of alendronate for thetreatment of osteoporosis (See Colon-Emeric, C. S., JAMA, 2006, 296(24):p. 2968-9; Ma, Y. L., et al., Endocrinology, 2003, 144(5): p. 2008-15).As shown in FIG. 9, two days after the i.v. injections, LLP2A aloneincreased the number of proliferating cell populations within the marrowcavity. In contrast and as shown in FIG. 9, LLP2A-Ale increased theseproliferating cell populations adjacent to bone surface. In order tomonitor bone microarchitectural changes that resulted from thetreatments, repeated in vivo microCT of the distal femurs were performedjust prior to the first injections and after four weeks when the micewere sacrificed. As shown in FIG. 4, compared to the approximately 2-5%increase in cancellous bone mass in the PBS, LLP2A, and Ale-treatedgroups, LLP2A-Ale, at higher dose level (250 ng), increased cancellousbone mass by nearly 20%. As shown in FIG. 4, trabecular thickness wasincreased in two dose levels compared to the PBS-treated group. As shownin FIG. 5 a-c, LLP2A-Ale did not change early osteoblast maturation(P1NP levels) but increased osteoblast function (osteocalcin) withsignificant increases in surface-based bone formation parametersincluding osteoblast surface, mineral apposition rate and surface-basedbone formation rate at the distal femur metaphysis. As shown in FIG. 6,LLP2A increased bone formation rate at both the endocortical andperiosteal surfaces of the mid-femurs (p>0.05). As shown in FIG. 6,LLP2A significantly increased bone formation rate at both theendocortical and periosteal surfaces of the mid-femurs (p<0.05). Therewere no dose-responses observed at the cortical bone compartment.

In FIG. 9, Two-month-old female 120SvJ mice received a singleintravenous injection of PBS, alendronate (500 ng/mouse), LLP2A (5nm/mouse) or LLP2A-Ale (10 ng or 500 ng/mouse). Mice were sacrificed 48hours after the treatment.

MicroCT.

The right distal femur from each of the animals was scanned usingMicroCT (VivaCT 40, Scanco Medical, Bassersdorf, Switzerland), with anisotropic resolution of 10 μm in all three spatial dimensions. The scanwas initiated at the lateral periosteal margin through the medialperiosteal margin of the distal femur and 0.1 mm from the highest partof the growth plate continuing proximally for 2 mm. Mineralized bone wasseparated from the bone marrow with a matching cube 3D segmentationalgorithm. A normalized index, trabecular bone volume/total volume(BV/TV), was utilized to compare samples of varying size. Trabecularthickness (Tb. Th), trabecular separation (Tb.Sp) and trabecular number(Tb.N) were also calculated as described previously (Lane, N. E. et al.,J Bone Miner Res, 2006, 21(3): p. 466-76; Yao, W., et al., ArthritisRheum, 2008, 58(6): p. 1674-86; Yao, W., et al., Arthritis Rheum, 2008,58(11): p. 3485-3497).

Bone Histomorphometry.

After fixation in 4% paraformaldehyde, the right distal femurs,mid-femurs and the 5^(th) lumbar vertebral bodies were dehydrated ingraded concentrations of ethanol and xylene and embedded un-decalcifiedin methyl methacrylate. The frontal sections (8 μm thick) were cut usinga vertical bed microtome (Leica/Jung 2265) and affixed to slides coatedwith a 2% gelatin solution. Unstained 8-μm-thick sections were used forassessing fluorochrome labeling and dynamic changes in bone. Bonehistomorphometry was performed using a semi-automatic image analysis(Bioquant Image Analysis Corporation, Nashville, Tenn.) linked to amicroscope equipped with transmitted and fluorescent light. Boneturnover measurements included single- (sL.Pm) and double-labeledperimeter (dL.Pm), interlabel width (Ir.L.Wi) and osteoclast surface.These indices were used to calculate Mineralizing Surface (MS/BS) andmineral apposition rate (MAR) and surface-based bone formation rate(BFR/BS) at the trabecular, endocortical and periosteal surfaces (Lane,N. E., et al., J Bone Miner Res, 2006, 21(3): p. 466-76; Yao, W., etal., Arthritis Rheum, 2008, 58(6): p. 1674-86; Yao, W., et al.,Arthritis Rheum, 2008, 58(11): p. 3485-3497; Yao, W., et al., Bone,1999. 25(6): p. 697-702).

Immunohistochemistry Staining.

Bone samples were fixed in 4% paraformaldehyde, decalcified in 10% EDTAfor 10 days and embedded in paraffin. Four-μm sections were obtained andincubated in 3% H₂O₂ in water to block endogenous peroxidases. Then theslides were incubated with 1% normal donkey serum in PBS Tween 20 inPBS. Then the sections were incubated with the primary antibodies. Afterblocking with peroxidase, the sections are incubated with the secondaryantibody for detection. Negative control was included where primaryantibody was omitted to differentiate unspecific staining.

Histochemical Analyses of Enzyme Activity.

Following sacrifice, small portions of organs were harvested and frozenin Optimal Cutting Temperature embedding media (Sakura, Torrance,Calif.) and sectioned in 12 μm thick slices. GUSB-specific histochemicalanalysis was performed using naphthol-AS-BI-β-D-glucuronide(Sigma-Aldrich) as a substrate, followed by counterstaining with methylgreen.

Statistics.

Statistical significance was assessed by analysis of variance followedby Dunnett's post-hoc test for comparisons between the treatment groupsto the PBS vehicle control group. P<0.05 was considered significant. Allvalues are expressed as means±standard deviation.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, one of skill in the art will appreciate that certainchanges and modifications may be practiced within the scope of theappended claims. In addition, each reference provided herein isincorporated by reference in its entirety to the same extent as if eachreference was individually incorporated by reference. Where a conflictexists between the instant application and a reference provided herein,the instant application shall dominate.

What is claimed is:
 1. A compound of Formula I:

wherein each R¹ and R² is independently selected from the groupconsisting of halogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, and C₁₋₆ haloalkyl; R³is selected from the group consisting of H, C₁₋₆ alkyl and C₃₋₈cycloalkyl; X is selected from the group consisting of O, S and NH; Y isselected from the group consisting of O and NH; Z is a peptide having3-20 independently selected amino acids, wherein at least one amino acidis selected from the group consisting of an unnatural amino acid and aD-amino acid; L is a linker D is a hisphosphonate drug; subscripts m, nand q are each independently from 0 to 2; and salts and isomers thereof.2. The compound of claim 1, which is selected from the group consistingof:


3. The compound of claim 1, having the formula:


4. The compound of claim 1, wherein peptide Z has the formula:-X_(AA1)-X_(AA2)-X_(AA3)-(X_(AA4))_(p)- wherein X_(AA1) is selected fromthe group consisting of a hydrophobic amino acid and derivatives oflysine, homolysine (Hly), ornithine (Orn) and α,γ-diaminobutyric acid(Dbu); X_(AA2) is a negatively charged amino acid; X_(AA3) is ahydrophobic amino acid; X_(AA4) is selected from the group consisting ofa naturally-occurring amino acid, an unnatural amino acid, and a D-aminoacid; and subscript p is 0 or
 1. 5. The compound of claim 4, whereinX_(AA1) is lysine-A38 (Lys38); X_(AA2) is α-aminohexanedioic acid (Aad);X_(AA) is a D-amino acid; and subscript p is
 0. 6. The compound of claim1, wherein peptide Z is selected from the group consisting of-Lys38-Aad-D-Phe, -Lys38-Aad-Ach, -Lys38-Aad-D-Nal-2, -Lys38-Aad-Ile,-Lys38-Aad-Val, and -Lys38-Aad-Leu.
 7. The compound of claim 1, whereinpeptide Z is -Lys38-Aad-Ach.
 8. The compound of claim 1, wherein tinkerL comprises at least one of N-(8-amino-3,6-dioxa-octyl)succinamic acid(EBES) and polyethylene glycol (PEG).
 9. The compound of claim 1,wherein linker L is selected from the group consisting of:

wherein k is from 0 to
 6. 10. The compound of claim 1, wherein D has theformula:

wherein R⁴ is selected from the group consisting of H, OH and halogen;R⁵ is selected from the group consisting of H and C₁₋₆ alkyl; andsubscript t is from 1 to
 6. 11. The compound of claim 1, wherein D hasthe formula selected from the group consisting of:


12. The compound of claim 1, having the formula selected from the groupconsisting of:


13. A pharmaceutical composition comprising a compound of claim 1 and apharmaceutically acceptable excipient.
 14. A method of treatingosteoporosis, comprising administering to a subject in need thereof, atherapeutically effective amount of the compound of claim
 1. 15. Amethod of promoting bone growth, comprising administering to a subjectin need thereof, a therapeutically effective amount of the compound ofclaim
 1. 16. A method of treating low bone mass, comprisingadministering to a subject in need thereof, a therapeutically effectiveamount of the compound of claim
 1. 17. A method of treating secondaryinduced low bone mass in a disease or condition, comprisingadministering to a subject in need thereof a therapeutically effectiveamount of the compound of claim 1, wherein the disease or condition isarthritis, immunosuppressant induced low bone mass, or chemotherapyinduced low bone mass.
 18. A method of treating a bone injury,comprising administering to a subject in need thereof a therapeuticallyeffective amount of the compound of claim
 1. 19. The method of claim 18,wherein the bone injury is a fracture.
 20. A method of treatingsecondary induced low bone mass in a disease selected from the groupconsisting of rheumatoid arthritis, juvenile rheumatoid arthritis,ankylosing spondylitis, celiac disease, inflammatory bowel disease, andulcerative colitis, comprising administering to a subject in needthereof a therapeutically effective amount of the compound of claim 1.21. A method of treating steroid induced osteoporosis, comprisingadministering to a subject in need thereof a therapeutically effectiveamount of the compound of claim
 1. 22. The method of any of claims14-16, 17, 18-19, or 21, further comprising administering to the subjectmesenchymal stem cells with the compound of claim 1.